cellutions 2012v4

7/29/2019 Cellutions 2012V4

1/32

Cellutions

Vol 4: 2012The Newsletter orCell Biology Researchers

Take control.

A novel RNA detection reagent enables gene

expression analysis in living cells Page 9

CellASIC ONIX live cell imaging platorm or

neural stem cell microenvironment control Page 13

Assessing enriched murine CD4+ T cells

dierentiated towards eector T helper cell lineages

with the Scepter 2.0 handheld automated cell

counter Page 18

Product Review Autophagy: Mechanisms

and connections to apoptosis Page 26

To subscribe to the quarterly Cellutions newsletter,

please visitwww.millipore.com/cellquarterlynews

Microuidic perusion enables long-term cell

culture, precise microenvironment control and

gene expression analysis Page 3

EMD Millipore is a division o Merck KGaA, Darmstadt, Germany
http://www.millipore.com/?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/reply/form/cellutions?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/reply/form/cellutions?cid=BIOS-C-EPDF-1007-1301-BS


2/32

PRODUCT HIGHLIGHT

Take control.Design dynamic experimentsthe CellASIC way.

Biology is so much more than DMEM/FBS, 37 C, 5% CO2.

Model experiments creatively and achieve true cultureconditions with the CellASIC ONIX Microuidic Platorm.Push the boundaries o your cell biology experiments in an

in vivo-like environment its easy to program automatedchanges to conditions and track cell responses with thisexible, intuitive platorm.

Read the articles on page 3 and page 13 o this issue.

The power is yours.www.millipore.com/CellASIC
http://www.millipore.com/?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/life_sciences/flx4/cellasic_live_cell_imaging?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/life_sciences/flx4/cellasic_live_cell_imaging?cid=BIOS-C-EPDF-1007-1301-BS


3/32

Microuidic perusion enableslong-term cell culture, precisemicroenvironment control

and gene expression analysisPaul Hung, Cindy Chen, Philip Lee, Terry Gaige, and Alex Mok

EMD Millipore Corporation

IntroductionThe analysis o living cells in vitrois critical to

understanding basic biology, signaling pathways,

drug eects, and disease models. Current methods

provide excellent means to interrogate living cells

via biomolecular probes, uorescence microscopy,and genetic manipulation1. However, technology or

environment control o living cells during analysis has

not advanced signifcantly since the Petri dish. There is

a growing body o evidence to indicate that the cellular

environment, or niche, is just as important (or even

more critical) than genetic actors or determining cell

phenotype2. Thereore, a method or providing more

accurate, dynamic control o living cells has the potential

to dramatically advance the state-o-the-art or live cell

analysis3,4.

The CellASIC ONIX Microuidic Platorm, in conjunction

with the CellASIC ONIX Microuidic Plate, provides

perusion-based microenvironment control or long-

term, high quality, live cell microscopy (Figure 1). The

microuidic chamber recreates the physiologic mass

transport condition or optimized cell health. Four

upstream uidic channels allow controlled exposure o

the cells to dierent solutions during live imaging. The

plate can also be cultured in a standard incubator using

a dedicated gravity driven ow channel. The cells are

in contact with a #1.5 thickness (170 m) optical glass

surace, enabling high quality imaging using an inverted

microscope. An integrated microincubator system delivers

temperature and gas control to the microuidic chambers.

In this study, we used the M04S Microuidic Plate

to demonstrate long-term culture o adherent cells,

to create dynamic solution profles (media switching

and spatial gradient), to immunostain cells within the

microuidic chamber, and to analyze gene expression.

Because gene expression is inuenced by numerous

cell cultural parameters such as soluble biochemical

actors, extracellular matrices, and stability o the

microenvironment, accuracy and physiological relevance

o gene expression analysis can be enhanced by

perorming such analyses in a dynamically controlled,

bio-inspired, microuidic system. Thereore, we

demonstrate here the ability to conduct gene expression

analysis using the CellASIC ONIX Microuidic Platorm

with M04S Microuidic Plates. Ultimately, directly

correlating gene expression patterns to phenotypes

observed during live cell imaging can provide powerul,

meaningul unctional genomics data, revealing

signaling networks and novel biomolecular interactions.Using breast cancer cell line MCF10A, we quantifed

the expression o Epidermal Growth Factor Receptor

(EGFR) using standard quantitative reverse transcription

polymerase chain reaction (qRT-PCR) techniques.

A BFigure 1.

Cells cultured in theM04S microuidicchamber. (A) HT-1080

cells imaged underphase contrast and (B)immunostained or nuclei(blue), actin (red), andmicrotubules (green).Fixing and staining wereperormed within themicrouidic chamber.Images were acquiredwith a 40X objective lens.


4/32

Materials and MethodsThe CellASIC ONIX Microuidic Platorm

The M04S Microuidic Plate has a 96-well ootprint to

ft to typical microscope stage holders. The microuidic

chamber (our in each plate) has been designed to be

compatible with standard culture methods, including

surace coating, 3D culture, and co-culture. The bottom

surace o the chamber is plasma-activated glass, which

supports most standard cell lines without coating,

including HeLa, NIH3T3, MCF7, MCF-10A, HT1080,

HUVEC, and others.

The custom well layout maximizes live cell imaging

capabilities. The M04S has our independent ow units

(A-D), with each unit containing eight inlet and outlet

wells (one gravity inlet, our switching inlets, one cell

inlet, two waste outlets). The our cell culture chambers

are centralized under a single large imaging window

(Figure 2). The chamber-to-chamber distance is

5.2 mm, reducing objective travel time and ocus drit.The bottom surace o the plate is a #1.5 thickness

(170 m) optical glass slide to maximize quality o high

resolution, high numerical aperture imaging. The plate

houses all experiment solutions, allowing control with

an external pneumatic maniold (Figure 3). The maniold

lets the user direct ow rates and select exposure

solutions without perturbing the microscope stage. A

gas line allows control o the environment within the

microchambers through a network o gas-permeable air

diusion channels. Temperature is regulated through an

on-board heater/chiller on the maniold.

Figure 3.

Side view schematic o the microuidic plate with micro-incubation maniold on a microscope stage. The bottomsurace o the microuidic plate is a thin glass sheet, allowinghigh quality cell imaging. The plate is sealed to a pneumaticmaniold, allowing user control o the ow profle duringimaging. Additional air channels allow control o the gasenvironment.

Figure 2.

The M04S plate contains our independent ow units (A-D),each with our upstream solution inlets, a gravity ow inlet,a cell inlet, and two waste wells. The culture chamber is2.8 mm in diameter (120 m height) and is surrounded witha microabricated perusion barrier. Inlet 1 is a gravity owwell, allowing long term cell culture in a standard incubatorwithout a pressure system. Continuous ow o solutions rominlets 2-5 creates a dynamic exposure profle during live cellimaging.

A

D

C

B

1 765432 8

5.2 mm

Imaging Window

Flow Inlets (2,3)

Cell Inlet (6)Cell Culture Chamber

(2.8 mm diameter)Waste(7-8)

PerfusionBarriers

Unit Marker

Air Channels

Gravity Inlet (1)

Flow Inlets (4,5)

Gravity Inlet (1)

Gas-permeablemembrane

Cells

Recirculatinggas mixture

Manifold

Air pressure-driven flow

Convective Peltierheat exchanger

#1.5 thickness(170 m) glass

Glass window

MicrofluidicPlate


5/32

Cell loading

Cells are loaded into the culture chamber using a capillary-

driven method. This allows the user to load cells using a

pipette, and can be done in a sterile laminar ow hood

without any external systems. The cell suspension is

pipetted into the cell inlet well (6) and liquid is aspirated

out o the waste wells (7 and 8). This creates a surace

tension orce that pulls the cell suspension into the

chamber. As the cells enter the chamber, the ow profle

allows them to settle to the oor without any stress.

Typically, loading into the microchambers occurs within

3 minutes and cells are allowed to settle or up to 30

minutes. I more cells are desired, higher concentrations

(or repeated loading cycles) can be implemented (Figure 4).

Figure 4.

Cell loading in the microuidic chamber. (A) Each chamber canbe seeded with up to a ew thousand cells by varying the celldensity during capillary ow loading. Images taken with a 4Xobjective o HT-1080 cells loaded at (B) 0.5, (C) 1.0, (D) 2.0,and (E) 4.0 million cells/mL.

Ater the cells settle to the bottom o the chamber,

continuous ow o medium enables them to attach and

grow. The chamber surace can be coated prior to cell

loading by owing the coating solution (e.g., poly-D-

lysine, collagen, fbronectin) into the chamber using

the same method. Alternatively, three-dimensional (3D)

culture can be achieved by mixing the cell suspension

with a gel matrix and loading together, allowing thecells/gel to polymerize within the chamber. The design

o the ow channels allows continuous perusion even

in the presence o 3D gel.

Gene expression analysis

Non-neoplastic human mammary MCF-10A epithelial

cells (obtained rom ATCC (CRL-10317) ) were grown in

Dulbeccos Modifed Eagle Medium F-12 (DMEM/F-12)

supplemented with 2% horse serum (Gibco), 5 ng/mL EGF

(Invitrogen), 0.5 g/mL hydrocortisone (Sigma), 100 ng/mL

cholera toxin (Sigma) and 10 g/mL insulin (Gibco) ina 5% CO

2atmosphere at 37 C. Prior to cell loading,

MCF-10A cells were prepared in a cell suspension o 1 X

106 cells/mL in the growth medium. Upon opening the

M04S microuidic plate packaging, priming solutions

were aspirated rom medium inlet well 1, cell inlet

well 6, medium outlet well 7 and cell outlet well 8.

Ater 10 L o cell suspension was loaded into well

6, medium in well 7 was aspirated again to distribute

cells evenly by capillary action. Plates were then laid

at in the incubator or an hour which allowed cells

to settle without any ow disturbance. Ater one hour

o incubation, 300 L o growth medium was added to

medium inlet well 1 and 50 L o the culture medium

into well 7 to initiate gravity driven perusion o growth

media during cell culture. Plates were then placed in the

incubator until cells were conuent.

To lyse the cells, medium was aspirated rom medium

inlet well 1, medium outlet well 7 and 8, ollowed by

pipetting 50 L o 1X PBS (Cellgro) into well 1. Ater

sealing the plate to the maniold, the CellASIC ONIX

FG Sotware was used to apply 4 psi on well 1 or 1

minute to rinse the channels and wash out cell debris inthe chambers. The maniold was then unsealed and all

liquid in well 1, 7, and 8 was aspirated. Then, 50 L o the

RNA extraction RNeasy RLT buer (Qiagen) was added

to well 1. Ater sealing the plate to the maniold, a cell

lysing program was initiated on the CellASIC ONIX FG

Sotware (4 psi on well 1 and 0.25 psi on well 6 or 20

seconds). We then unsealed the maniold and collected

20 L o the cell lysates rom well 7. RNA was extracted

using the RNeasy mini kit (Qiagen), ollowing the RNA

extraction procedures instructed in the manuacturers

manual.

B

A

D

C

E

4000

3000

No.

Cellsloaded

Cell Suspension (106 cells/mL)

2000

1000

29

0 1 2 3 4


6/32

Total RNA was then synthesized into cDNA using

iScript cDNA synthesis kit (Bio-Rad) with random

hexamer primers. Quantitative PCR was perormed

with the ollowing primers: EGFR orward

(5- GGCAGGAGTCATGGGAGAA-3) and reverse

(5-GCGATGGACGGGATCTTAG-3). Samples were

normalized using oligonucleotides specifc to

RNA encoding the house-keeping gene, GAPDH:

orward (5-ACCCACTCCTCCACCTTTG-3) and reverse(5-CTCTTGTGCTCTTGCTGGG-3). cDNA was diluted 1:25

or GAPDH and 1:2 or EGFR beore qPCR. Quantitative

PCR was perormed using SsoFast Evagreen Supermix

(Bio-Rad) on the MiniOpticon Real-Time PCR Detection

System (Bio-Rad). The conditions or PCR were 95C,

3 minutes ollowed by 40 cycles o 95C 5 seconds, 60C

10 seconds. Analysis variance and post hoc tests o

quantitative PCR results were carried out using Bio-Rad

MiniOpticon sotware.

ResultsSolution switching

A key eature o the M04S plate design is that solutions

can be changed during live cell imaging without

perturbing the plate or microscope. This enables tracking

o cell responses to changing solution environments.

The M04S allows our dierent solutions to be switched

during the course o the experiment.

Exposure solutions are introduced rom the our inlet wells

and ow through the chambers to the waste (well 7). Wells

7 and 8 are connected as a shared outlet or increased

volume. Well 1 is a gravity ow well, with a perusion

rate o approximately 80 L/day. This is used or pre-

culture o cells in the M04S plate in a standard incubator

when solution exchange or imaging is not necessary; or

example, to expand or dierentiate cells over a period o

a ew days. Wells 2-5 are the pressure-driven wells. Theow rate and exchange times are given in Figure 5. The

highly laminar ow profle means that when the input

solution is changed, a sharp uid interace is created

that moves across the culture area rom let to right. The

velocity o this ront is given in Figure 5A. The actual local

exchange time (the transition rom solution 1 to 2 around

the cells) happens quickly, typically in a ew seconds. The

small volume o the culture chamber enables ast solution

exchange at ow rates rom 5-80 L/hour. This means that

a typical imaging experiment (with 300 L per inlet well)

can run or up to 72 hours.

As one example o ow switching, two solutions

(phosphate-buered saline (PBS) and dextran-conjugated

uorescein, 3 kDa, Invitrogen) were switched at 10-minute

intervals (Figure 5B). Note the rapid and complete response

o the solution, creating a clean step unction in the

culture region. Since all our channels converge near the

culture chamber, the M04S plate minimizes the dead

volume during switching to a ew nanoliters.

In addition to ow switching, the CellASIC ONIX

Microuidic Platorm was used to create spatial

gradients across a chamber (Figure 5C). When more

than one channel was owed simultaneously, laminar

ow and diusion across the interace created a stable

spatial gradient. For sensitive kinetic experiments, it is

recommended that a tracer dye be used to accurately

ollow solution ow profles.

Figure 5.

A) Flow rate through the chamber as a unction o pressure applied by the CellASIC ONIX Microuidic Platorm to the inlet wells (V2 V5) o M04S plate. Theow rates allow long-term, continuous ow experiments to be perormed on the microscope stage. The inlet wells hold 350 L o solution, allowing a singleexperiment to run or up to 72 hours. In general, a ow pressure o 1 psi is suitable or nutrient exchange. (B) Sequential switching between uorescent dye andPBS. Intensity was measured in the center o the culture region every minute with ow at 2 psi rom 2 channels. Data was plotted normalized to the max/min ointensity images. (C) Spatial gradient in the M04S chamber o uorescein conjugated dextran (3kDa). Intensity was measured in the y-direction o the chamber.

Light green line shows ow rom 2 channels simultaneously o 0% and 100% dye at 0.5 psi each. Dark green line shows ow rom 4 channels simultaneouslyat 0%, 25%, 50%, and 100% dye at 0.5 psi each. Di usion across the interaces creates a smoothed profle. Flow at aster rates will lead to sharper, step-likeboundaries.

A

Pressure (psi)

FlowR

ate(L/hr)

Wells 2-5

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150160

170

180

0 1 2 3 4 5 6 7 8 9

1.0

0.8

0.6

0.4

0.2

00 20 40 60

Minutes

B

Intensity

1.0

0.8

0.6

0.4

0.2

00 400 800 1200 1600

C

Microns (y-direction)

NormalizedIntensity


7/32

Long-term cell culture

MDA-MB-231 cells were grown using the CellASIC

ONIX Microuidic Platorm in M04S perusion plates or

three days with continuous perusion. Figure 6, showing

the cells at our dierent time points, illustrates that cell

health and morphology were preserved or the entire

length o the experiment.

Time lapse imagingThe avorable cell culture environment in the M04S

chamber allows long term maintenance o adherent cells

under well-controlled conditions. This enables enhanced

live cell tracking o cellular events in response to changes

in media, cell cycle, drug dosing, and other stimuli. We

demonstrated two common methods or high resolution

cell analysis using microscopy: 1) immunostaining, and

2) transection. To acilitate immunostaining o cells

cultured in the M04S plate, an automated ow protocol

was set up to sequence fxing, permeabilizing, blocking,

primary antibody, secondary antibody, and all washsteps. This enabled monitoring o live cells in the M04S

plate, and then subsequently fxing the same cells and

analyzing by immunouorescence.

In a second example, live cells cultured in the M04S plate

were transected with GFP-tubulin (Invitrogen Cellular

Lights reagent). Ater exposure to the GFP reagent, the

cells expressed the protein o interest and could continue

to be tracked in the microuidic system (Figure 7).

Figure 7.On-chip staining o cells cultured in the M04S chamber. (A) HeLa cells cultured in themicrouidic chamber immunostained via ow exposure. Actin is shown in red, tubulin is shownin green. (B) MCF-10A cell cultured in the microuidic chamber and transected with GFP-tubulin via ow exposure. Images taken with a 100X objective lens.

A B

Figure 6.

Growth o MDA-MB-231 cells in the microuidic chamber withcontinuous perusion ater (A) 1 hour, (B) 1 day, (C) 2 days, and(D) 3 days. Images were acquired with a 4X objective lens.

A

C

B

D

Figure 8.

Images o the cell culture chamber beore (let) and ater (right) cell lysis. The cells in the imageat let were allowed to grow to conuency.

Gene expression analysisWe frst quantifed the total number o MCF-10A cells

cultured to semi-conuency in each chamber o the

our-chamber M04S plate using microscopy and Image

J sotware (http://rsbweb.nih.gov/ij/). Ater counting the

cells, we used an in-well lysis procedure and then removed

the lysates or analysis. To demonstrate the efciency

o the lysis protocol, we grew cells to near conuency in

the M04S Microuidic Plate and imaged the cell culture

chamber beore and ater lysis (Figure 8). The clear

chamber ater lysis indicated high collection efciency,

with no remaining cells let over in the chamber.


8/32

Figure 9.

(Top) Per-cell EGFR expression normalized to GAPDHexpression and total number o cells per chamber. (Bottom)Comparison o per-cell EGFR expression o MCF-10A cellscultured in the CellASIC ONIX Platorm (M04S plate) vs. instandard Petri dish.

Per Cell EGFR Expression(Microfluidics)

0.35

0.30

0.25

0.20

0.15

0.10

0.05

0.00

A B C D

Chambers

Norma

lizedExpressionValues

Per Cell EGFR Expression(Microfluidics vs. Petri Dish)

0.35

0.30

0.25

0.20

0.15

0.10

0.05

0.00

Dish Microfluidics

NormalizedE

xpressionValues

SummaryThe ability to control and monitor living cells is critical or understanding signaling

networks and complex phenotypes in response to stimuli. We have developed the

innovative CellASIC ONIX Microuidic Platorm using the M04S perusion chamber

plate to optimize cell microenvironment control while acilitating long-term, high

quality microscopy. This design has been demonstrated with a wide range o cell lines

or uorescence quantifcation, solution exchange response, and time-varying inputs.

Existing cell analysis methods such as immunostaining, transection, uorescent

probes, and more, are easily adapted to the microuidic ormat. Further, the ease o

use, exibility, and accessibility o this advanced technology platorm should prove

benefcial to a wide range o cell biology applications, including accurate, meaningulgene expression analysis.

Reerences1. Wessels JT, et al. Advances in cellular, subcellular, and nanoscale imaging in vitroand in vivo.

Cytometry A. 2010 Jul;77(7):667-76.

2. Bissell MJ, Radisky D. Putting tumours in context.

Nat Rev Cancer. 2001 October;1(1):46-54.

3. Lee P, Gaige T, Hung P. Microluidic systems or live cell imaging. Methods Cell Biol.

2011;102:77-103.

4. Lee PJ, Gaige TA, Hung PJ. Dynamic cell culture: a microluidic unction generator or live cell

microscopy. Lab Chip 2009 Jan 7;9(1):164-6.

Description Catalogue No.

CellASIC ONIX Microuidic System Package includes CellASIC ONIX Microuidic System, Maniold,

Accessory Box, and CellASIC ONIX FG Sotware EV262

CellASIC ONIX Microincubator Package or Temperature and Gas Control: Includes CellASIC ONIX

Microincubator Controller, Microincubator Maniold, and Accessory BoxMIC230

M04S Microuidic Switching Plate or Mammalian Cells (4 Chambers) M04S-03-5PK

Available romwww.millipore.com.

FEATURED PRODUCTS

By conducting RT-PCR analysis o the collected lysates, we quantifed EGFR and GAPDH

gene expression in the cells. The EGFR expression per cell was normalized to GAPDH and

total cell number in each chamber (Figure 9, top). The same experiment was perormed

on cells cultured on standard petri dishes. We ound an almost 3X dierence in per cell

EGFR expression between the M04S plate and a standard petri dish (Figure 9, bottom).

While we do not know the cause o this increase, it was highly reproducible and may

reect the environmental conditions supported in the microuidic chambers.
http://www.millipore.com/catalogue/item/EV262?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/MIC230?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/M04S-03-5PK?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/M04S-03-5PK?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/MIC230?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/EV262?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/M04S-03-5PK?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/MIC230?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/EV262?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/?cid=BIOS-C-EPDF-1007-1301-BS


9/32

A novel RNA detection reagentenables gene expression analysisin living cellsDon Weldon, Erika Wells, James Graham, Laura White, Kevin Su, Alex Ko,

Yuko Williams, Martin Santillan, Jessica OConnor and Matthew Hsu


IntroductionDetecting gene expression has traditionally been limited

to technologies that examine expression in lysed or fxed

cell populations. The ability to detect RNAs in individual,

live cells can enable an unequivocal assessment o gene

expression changes that occur in direct response tospecifed perturbations. Determining which genes are

up- or down-regulated in these perturbed cells provides

insight into the relationships between gene expression

networks and cell unctions.

We present the SmartFlare detection reagent

(Figure 1), a novel probe capable o detecting specifc

mRNAs and miRNAs in live, intact cells1. This technology

allows or carrier-ree cellular endocytosis o the

reagent, ollowed by detection and relative quantitative

analysis o RNA levels. Because the reagent leaves thecell ater the detection event, the same sample can

be used or any downstream analysis, enabling the

measurement o multiple biomarkers or downstream

unctionalities in the same cells. Additionally, this

reagent requires no upront sample preparation, has no

toxic eects on cellular ate and no known nonspecifc,

o-target eects. Compared to currently used methods

or interrogating RNA that involve examination o

non-native, amplifed RNA targets, SmartFlare probes

have the potential to provide results that show greater

correlation to in vivoobservations. In vivo, most cells

reside in heterogeneous tissues, and cell-to-cell variation

in gene expression can be subtle yet crucial or tissueunction. SmartFlare technology can be used to

quantitate RNAs in individual cells, providing heretoore

unobtainable gene expression inormation that

distinguishes one heterogeneous cell population rom

another with high resolution.

Figure 1.

SmartFlare RNA Detection Probe

Gold-quencheddye on reporterstrand

Goldnanoparticle

Capture strand/reporter strandduplex


10/32

Materials and Methods

Figure 2 shows the structure o a SmartFlare probe and

its mechanism o action. Each probe consists o a gold

nanoparticle conjugated to many copies o the same

double stranded oligonucleotide encoding the target

sequence. One strand o the oligonucleotide bears a

uorophore that is quenched by its proximity to the gold

core. When the nanoparticle comes into contact withits target, the target RNA displaces the uorophore. The

reporter strand, now unquenched, uoresces and can be

detected using any uorescence detection platorm.

Wavelength (nm)

Intensity

(C

PS)

Cy5 fluorescence (probe + target-specific oligo)

Cy5 fluorescence (probe + nontarget oligo)

Cy5 fluorescence (probe alone)

0

50000

150000

650 700 750

100000

Figure 3.

Specifc detection otarget RNA sequences.

The SmartFlare probealone (in buer) generateda background level ouorescence (pink).Following addition oa noncomplementaryoligonucleotide, theuorescence signal (red)was unchanged. Uponaddition o a complementarysequence, the uorescencesignal (yellow) reached itsmaximum intensity.

Assay Protocol

The ollowing general protocol is used or SmartFlare

RNA Detection Probes; however, concentrations o

probe, incubation times and detection methods can vary

depending on the specifc cell types and probes used.

1. Culture cells to 60-80% conuency

2. Add SmartFlare probe3. Incubate overnight (16 hours)

4. Detect via ow cytometry, microscopy,plate reader, or any other uorescent

detection platorm

Results

As shown in Figure 3, a typical SmartFlare RNA

Detection Probe exhibits specifcity or its target, as

evidenced by the increase in uorescence upon addition

o the target sequence, and lack o signal when a non-

target sequence is added in equal amounts.

Figure 2.

Molecular mechanismo SmartFlare RNADetection Probe

The oligo duplexes are designed withan RNA capture sequence and a

complementary reporter sequence.

In absence o target, probeis quenched by gold and does

not uoresce

When Target is present it will bindthe capture strand, releasing

the reporter strand, which then uoresces.

Target

+

.

Capture Strand

Reporter Strand

.

-AAAAAAATCAACCATACACCGTGACTTTGCTTGACCC

-AGTTGGTATGTGGCACT


11/32

0

100 101 102 103 104

10

5

25

20

15

40

35

30

Count

miR-21 Cy3 (YEL-HLog)

HEK-293 DU-145

DU-145 HEK-293

Fluorescence(M5-510)

Cycles

1 2 6 9 10 1613 19 22 25 29 4034 37314

Fluorescence(M5-510)

Cycles

30

40

50

60

70

80

90

100

110

40

50

60

70

80

90

100

110

1 2 6 9 10 1613 19 22 25 29 4034 37314

MCF-7No probe HeLa

M CF-7 HeLa

0

20

100 101 102 103 104

40

60

80

100

120

140

EGFR mRNA probe (RED2-HLog)

FGF2 mRNA probe (RED2-HLog)

Count

No probe HT1376

0

100 101 102 103 104

40

20

60

80

100

120

140

160

Count

HUVEC

HT1376 HUVEC

180

Sample application: miRNA quantitationto distinguish two cell types

Because miRNAs are potent regulators o gene

expression, requently determining cell ate, they can be

eective biomarkers or disease states. Cell types can

requently be distinguished comparing relative levels o

specifc miRNAs. A probe to miR-21 was developed to

test the ability o SmartFlare technology, in conjunctionwith ow cytometry, to detect dierences in expression

between two cell types: HEK-293 cells (expressing low

levels o miR-21) and DU-145 cells (expressing high

levels o mIR-21). Low expression was indeed seen in the

HEK-293 cells (Figure 4, ar let section o the histogram)

and DU-145 cells showed a distinctly higher expression

(Figure 4, ar right o the histogram).

SmartFlare quantitation correlates withresults rom quantitative PCR (qPCR)

By treating cells with SmartFlare probes and measuring

subsequent uorescence with ow cytometry, we

measured the levels o two mRNA targets, EGFR and

FGF2, in cell types that show dierences in expression

o each o these targets (Figure 5, let). To confrm

expression levels, we quantitated the same RNAs in these

cells using reverse transcription PCR (RT-PCR). Because

SmartFlare technology allows or downstream assays

ollowing detection and analysis, RT-PCR was perormed

on the same cells to compare the relative amounts o

target RNA in each cell type (Figure 5, right).

Figure 4.

Target-specifc miRNAdetection using owcytometry. Detection o amicro RNA target miR-21 inboth HEK-293 cells (typicallyused as a low expresser omiR-21) and in DU-145 (aprostate cancer cell lineknown to express high levelso miR-212) using owcytometry. The histogramshowing the HEK-293 cellpopulation is shown overlaidwith the histogram o theDU-145 cell population,reecting two distinctpopulations o cells based ontheir gene expression profle.

Figure 5.

Probe detection o mRNAlevels correlates to RT-PCRdata. Using SmartFlaretechnology to determine themRNA levels o EGFR mRNA(A) in HeLa and MCF-7 cellsas well as FGF2 mRNA (B)in HUVEC and HT1376 cells,both o which correlate totheir RT-PCR levels. Flowcytometry provides addedinormation at the singlecell level as well as how theexpression is distributedwithin the population.

As expected, the cells that yielded lower Ct values

correlated with higher gene expression by ow

cytometry, indicating that the higher the level o RNA

target present is reected in the histogram as having a

higher mean uorescence intensity.

The histograms revealed additional inormation on the

variation o the expression within each cell population.

For example, the HeLa cells (sharp light green peak)

showed much more uniorm expression o EGFR than

MCF-7 cells (wider dark green peak).

A.

B.


12/32

Fluorescence microscopy ollowingSmartFlare treatment reveals geneexpression dierences

Fluorescence microscopy was perormed on HeLa and

MCF-7 cells ollowing addition o either an EGFR probe or

a scrambled sequence (Figure 6). Both cell types showed

low levels o uorescence with scrambled sequence. The

HeLa cells showed a dramatic increase in uorescence with

the EGFR probe compared to the MCF-7 cells. The results

correlated with our ow cytometry and RT-PCR data.

be reused ollowing analysis and ater cell sorting or

additional downstream experiments on those same cells.

By enabling the researcher to discover cells that express

particular genes at particular levels in real time, thistechnology can enhance the signifcance o observed links

between genotype and phenotype in heterogeneous cell

populations, truly enhancing the value o data obtained

or RNA analysis.

Reerences1. Prigodich AE et al. Nano-Flares or mRNA Regulation and Detection.

ACS Nano. 2009 August 25; 3(8): 21472152.

2. Jinsong Li, MiR-21 Indicates Poor Prognosis in Tongue Squamous Cell

Carcinomas as an Apoptosis Inhibitor, Clin Cancer Res 2009; 15:3998

3. Prigodich AE et al. Multiplexed nanoares: mRNA detection in live cells.Anal Chem. 2012 Feb 21;84(4):2062-6.

Conclusions

We have shown that this novel live cell RNA detection

technology is easy to use and robust. These results

demonstrate that this reagent is sensitive and specifc

the SmartFlare probe emits uorescence only uponaddition o its complementary target sequence. It can

detect both micro and messenger RNA in the cytosol o

living cells and has shown good correlation to qRT-PCR.

Furthermore, the probes are platorm-agnostic, enabling

quantitation via ow cytometry or via microscopy,

without amplifcation.

The many potential uses o this breakthrough

technology include sorting o cells based on gene

expression (enabling even higher levels o enrichment

using additional intracellular markers), live celltracking o RNAs, and detection o multiple types o

biomolecules (such as protein and RNA) in the same

sample. Previous reports have already confrmed that this

technology can be used or multiplexed detection o up

to three dierent RNAs (using dierent uorophores),

enabling the normalization o gene expression levels

to housekeeping or control genes within individual

cells3. Notably, SmartFlare technology allows cells to

Scrambled Control

MCF-7Cells

HeLaCells

EGFR Target

Figure 6.

EGFR probe detection inhigh and low expressingcell lines. EGFR expressionin MCF7 and HeLa cellscompared to a scrambledsequence control. Specifcityor the target o interest is

evident in the uorescenceintensity o the EGFR probesin the HeLa cells vs theMCF-7 cells.


EGFR SmartFlare Probe SF-116

FGF2 SmartFlare Probe Coming soon

Scrambled Control Cy5 SmartFlare Probe SF-102

miR-21 SmartFlare Probe Coming soon

guava easyCyte 8HT Flow Cytometer 0500-4008


FEATURED PRODUCTS
http://www.millipore.com/catalogue/item/SF-116?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/SF-102?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/0500-4008?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/0500-4008?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/SF-102?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/SF-116?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/?cid=BIOS-C-EPDF-1007-1301-BS


13/32

AbstractNeural stem cells (NSCs) are sensitive to microenvi-

ronmental cues, including cell-cell contact, cell-ECM

interaction, nutrient and waste transport, as well as

environmental oxygen composition. However, how

these parameters in the microenvironment aect the

stem cells morphology, prolieration, and dierentia-

tion remains an open area or research. In this study,

we demonstrated how the CellASIC ONIX Microuidic

Platorm with its microuidic cell culture devices are

capable o multi-parametric microenvironment control

or NSC studies.

IntroductionNSCs are sel-renewable and multipotent cells that are

capable o generating various phenotypes o the nervous

system1

. First described in the subventricular zone othe adult mouse brain2, NSCs have received considerable

attention due to their potential or therapeutic use.

Research eorts generally divide into three ocus areas:

expansion, dierentiation and cell:cell interactions.

While commercial sources or NSCs have grown rapidly,

building a physiologically relevant in vitroNSC culture

model to thoroughly study the biology behind NSC

expansion, dierentiation and cell:cell interactions

remains challenging. In addition to two-dimensional

(2D) culture asks, researchers have attempted other

culture technologies such as three-dimensional (3D)

neurospheres3, scaolds4, and microuidics5. While each

technology has separately shown that NSCs are sensitive

to microenvironmental cues, there has been no unifed

platorm enabling researchers to systematically control

the microenvironment or NSC culture.

Figure 1.

(Above) CellASIC ONIX Microuidic Platorm with themicrouidic system, the microincubator controller andmicroincubator maniold. (Right) Layout o M04S plate(our independent units and eight wells per unit).

CellASIC ONIX live cell imagingplatorm or neural stem cellmicroenvironment controlPaul Hung, Cindy Chen, Philip Lee, Terry Gaige, Brandon Miller,

Andrew Zayac, Ivana Zubonja and Alex Mok



14/32

The CellASIC ONIX Microuidic Platorm oers

comprehensive cellular microenvironmental control or

cell culture studies (Figure 1). The ability to control

multiple parameters has been integrated into the

CellASIC ONIX Microuidic Platorm to enhance the

cellular microenvironment or NSC culture. To modulate

the degree o cell-cell contact, the system can be set up

to seed cells at varying density, leading to varying spatial

distribution. To enable changes to cell-ECM interaction,we developed protocols to coat the #1.5 coverglass

substrate with polyornithine/laminin, poly-D-lysine or

poly-L-lysine. For nutrient and waste transport, both

passive gravity-driven and pneumatically-driven control

can provide low-shear, steady-state perusion through

NSC cultures with solution switching. Since the plate

containing the cell culture chambers is made o

gas-permeable materials, it is possible to inoculate NSC

cell culture with dierent mixtures o gases. In this study,

we show that the oxygen microenvironment can be

tuned to range rom severe anoxia to hyperoxia. Tocharacterize NSCs grown in the microuidic-controlled

microenvironment, an automated immunostaining

protocol was developed to visualize nestin and Sox2

within NSC cell culture.

We also tested the speed at which gas conditions could

be changed using the microincubator, to assess the utility

o the system or studying cell responses to hypoxia.

The successul combination o environment control with

perusion culture in a microuidic platorm promises to

urther close the gap between in vitroexperiments and

in vivorelevance.

Materials and MethodsMaterials. Adult rat hippocampus neural stem cells and

antibodies recognizing Nestin and Sox2 were obtained

rom EMD Millipore (Cat. Nos. SCR021 and SCR022).

The CellASIC ONIX Microuidic System (Cat. No. EV262),

Microincubator Controller (Cat. No. MIC230), Tri-Gas

Mixer (Cat. No. GM230), and Microuidic Plates

(Cat. No. M04S-03-5PK) were also purchased rom

EMD Millipore. Poly-L-ornithine and laminin wereacquired rom Sigma. For poly-L-ornithine, the stock

solution was prepared with sterile, deionized water to a

fnal concentration o 50 g/mL. Laminin was prepared

with 1x phosphate-buered saline (PBS) at 7 g/mL.

The coating solutions were stored at 4 C. All other

reagents, such as rat neural stem cell culture medium

(Cat. No. SCM009) and basic fbroblast growth actor

(Cat. No. GF003) were obtained rom EMD Millipore. For

uorescent microscopy, an Olympus IX-71 inverted

uorescence microscope with an automated stage was

used. To validate the oxygen concentration in the cell

culture chamber, an oxygen sensor rom Presense was

attached inside the microuidic device.

Coating the cell culture chamber with extracellular

matrices. Liquid was aspirated rom wells 1, 6, 7, and 8

without aspirating liquid rom the inner rings (see user

guide or detailed protocol)6. 300 L o poly-L-ornithinesolution was then added to well 6. The plate was then

placed inside a traditional cell culture incubator at 37 C

or 24 hours to coat the M04S microchamber. Ater 24

hours, the plates were retrieved and liquid in wells 6, 7

and 8 was aspirated. 300 L o sterile, deionized water

was used to rinse the well beore another 300 L o

sterile water was added to well 6 to wash the

microchamber. The plate was then placed in an incubator

or 4 hours. For the second layer coating o laminin, the

water in wells 6 and 7 was aspirated. 300 L o the

laminin solution was then added to well 6. The plate wasreturned to the incubator or 24 hours. Beore seeding

the cells, the laminin solution in wells 6 and 7 was

aspirated. 300 L o 1x PBS was added to well 1 and

well 6. The device was then returned to the incubator or

2 hours to allow the PBS to gently wash out the

remaining laminin solution in the microuidic plate.

Cell seeding. The cell suspension was prepared

according to the vendors protocol and seeded at

both high and low densities. For low density seeding,

1 x 105/mL was recommended. We seeded cells at high

density (1 x 106/mL) to allow close cell-cell contact.

Beore introducing the cells into the microuidic device,

the liquid in well 1, 6 and 7 were frst aspirated. The inner

rings in well 6 and 7 were then careully aspirated

(extended aspiration will result in bubble introduction

into the microuidic channel). 10L o the cell

suspension (1,000 total cells or low cell density and

10,000 total cells or high cell density) was then added

into the inner ring o well 6. The plate was then placed in

a laminar cell culture hood or 30 minutes to allow the

cells to load into the microchamber and settle.

Gravity-driven perusion culture. To stabilize the cells,

300 L o the culture medium was added to well 1 to

allow gravity-driven perusion and the plate was placed

inside a regular incubator. For prolonged culture in the

incubator, the medium in wells 1 and 7 was aspirated

every 48 hours, and 300 L o the culture medium was

added into well 1 to re-establish gravity-driven perusion.

To culture the cells on the microscope, the PBS was

aspirated rom wells 2, 3, 4 and 5 but not the inner rings.

As a guideline, 300 L in each well, when owed at 0.5

psi, could provide ample nutrients to the cell cultures or

up to 36 hours.
http://www.millipore.com/catalogue/item/SCR021?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/SCR022?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/EV262?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/MIC230?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/M04S-03-5PK?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/SCM009?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/GF003?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/GF003?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/SCM009?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/SCR022?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/SCR021?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/M04S-03-5PK?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/MIC230?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/EV262?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/M04S-03-5PK?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/MIC230?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/EV262?cid=BIOS-C-EPDF-1007-1301-BS


15/32

Figure 3.

Brightfeld image o rat NSCs cultured in the CellASIC ONIX M04S Microuidic Plate onDay 8 (Let). Immunostaining o nestin (green) and Sox2 (red) indicated that these cellsremained undierentiated (Right).

Figure 2.

The microincubator controller provided precise control o gaseous microenvironment or themicrouidic cell culture. Gas exchange was accomplished within 15 minutes.

Automated immunostaining. 300 L PBS was

added to well 1 as the wash solution, 100 L o 4%

paraormaldehyde was added to well 2 as a fxing agent,

100 L o 0.2% BSA, 0.1% TritonX-100 in 1x PBS was

added to well 3 or permeation and blocking, 150 L o

primary antibody solution with 1% BSA was added to

well 4, and 100 L o secondary antibody was added to

well 5. To conduct the immunostaining automatically,

the pressure applied to each valve was programmedaccording to the ollowing parameters: V2 (fxing),

4 psi, 12 minutes; V1 (washing), 0.25 psi, 8 minutes;

V3 (permeation and blocking), 4 psi, 12 minutes; V1

(washing), 0.25 psi, 8 minutes; V4 (primary antibody),

4 psi, 60 minutes; V1 (washing), 0.25 psi, 8 minutes; V5

(secondary antibody), 4 psi, 40 minutes; V1 (washing),

0.25 psi, 5 minutes.

Testing NSC behavior with respect to gaseous

microenvironment. First, we determined the gaseous

microenvironment exchange rate. We purged the devicein pure nitrogen and let it stabilize or over 24 hours. A

pre-calibrated 5% CO2 gas was then introduced through

the microincubator. As depicted in Figure 2, the gas

exchanged thoroughly in about 15 minutes. To

investigate the impact o oxygen microenvironment on

NSC behavior, we seeded the cells under the normoxia

(20% oxygen) condition and let them stabilize in the

incubator or 24 hours. We then transerred the NSCs

onto three dierent microincubators and tri-gas mixers

tuned to three gas compositions buered with 5% CO2

and nitrogen: severe hypoxia (~0.1% O2), mild hypoxia

(~3% O2) and normoxia (~20% O2). Loading 300 L o the

culture medium in well 2, 3, 4 and 5, we were able

to culture the rat NSCs or up to our days, uninterrupted,

through pneumatic pumping o medium across the cell

culture chamber. Bright feld images were acquired every

day with a subset o cells immunostained or nestin

and Sox2.

ResultsThe key eatures o neural stem cells include sel renewal

and lack o dierentiation. To examine these signatures in

a gravity-driven perusion microuidic system over one

week, we used automated immunostaining to detect the

NSC markers, nestin and Sox2 (Figure 3). All the cells in the

chamber were successully stained, showing nestin in the

cytosol and Sox2 in the nucleus, which confrmed that the

rat NSCs maintained an undierentiated phenotype while

being cultured in this microuidic device.

6

5

4

3

%CO2

Time (minutes)

2

1

0

0:00:00 0:15:00 0:30:00 0:45:00 1:00:00 1:15:00 1:30:00 1:45:00


16/32

Figure 4.

Rat NSCs cultured at low densities under mildlyhypoxic conditions (top) and under normoxicconditions (bottom). Under hypoxic conditions,the cells seemed to disaggregate and spreadinto discrete cell bodies while under normoxicconditions, the cells tended to aggregate.

Figure 5.

High density seeding o NSCs in a microuidicchamber under conditions o mild hypoxia causesormation o neurospheres (top row, inset boxat 72 hours). Neurospheres were also imaged at

higher magnifcation (20X, bottom let). Whenstained or nestin and Sox2 (bottom right), onlythe outer layers o the neurospheres showed bothnestin (green) and Sox2 (red) expression; thecore displayed bright spots o Sox2 expression.This pattern was possibly caused by neurospherecompression by the ceiling o the microuidicchamber.

We ound that cells detached and washed away, presumably

due to cell death, 24 hours ater exposure to severe hypoxia

(data not shown). At low cell density, cells disaggregated

under mildly hypoxic conditions, which promoted single-

layer cellular growth. In contrast, under normoxic conditions,

the cells aggregated into multilayer cellular masses (Figure 4).

Due to the cell loading variations, the initial seeding density

(0 hour) in the normoxia chamber was higher than the mild

hypoxia chamber (data not shown).

To urther explore the eect o microchamber culture o rat

NSCs, we seeded the cells at high density under mildly

hypoxic conditions (3% O2). Ater our days, the cells ormed

neurospheres, though the spheres were compressed by being

confned by the ceiling o the microchamber. Staining or

nestin and Sox2 revealed that the core o this tightly packed

mass contained only bright spots o Sox2 while the outer

ring o the neurospheres exhibited both nestin and Sox2

expression (Figure 5).

24 hours 48 hours 72 hours

24 hours

3% 02

20% 02

48 hours 40X objective


17/32

Reerences1. Clarke, D. et al. Generalized Potential o Adult Neural Stem

Cells. Science 2000; 288 (5471): 1660-63.

2. Temple, S. Division and dierentiation o isolated CNS blast

cells in microculture. Nature 1989; 340: 471-73.

3. Serra M et al. J Neurosci Res. 2007 Dec;85(16):3557-66.

4. Lee YB et al. Exp Neurol. 2010 Jun;223(2):645-52.

5. Chung BG et al. Lab Chip. 2005 Apr;5(4):401-6.

6. EMD Millipore, User Guide: CellASIC ONIX M04S

Microluidic Plates. EMD Millipore Corporation, Billerica, MA

USA, 2012. Product Literature No. 00003670, Rev. A.

7. De Filippis L and Delia D. Hypoxia in the regulation o neural

stem cells. Cell Mol Lie Sci. 2011 Sep;68(17):2831-44.

8. Santilli G, et al. Mild hypoxia enhances prolieration and

multipotency o human neural stem cells. PLoS One 2009;

5(1):e8575

9. Takahashi K et al. Induction o pluripotent stem cells

rom adult human ibroblasts by deined actors. Cell 2007;

131(5):86187210. Storch A et al. Long-term prolieration and dopaminergic

dierentiation o human mesencephalic neural precursor

cells. Exp Neurol 2001; 170(2):317325

11. Aguirre A et al. Notch and EGFR pathway interaction

regulates neural stem cell number and sel-renewal. Nature.

2010 Sep 16;467(7313):323-7

12. Bakhru S et al. Direct and cell signaling-based, geometry-

induced neuronal dierentiation o neural stem cells.

Integr Biol (Camb). 2011 Dec;3(12):1207-14..

DiscussionThe cellular microenvironment, and the oxygen

microenvironment in particular, is known to regulate neural

stem cell metabolism, prolieration, surivival and ate7. Mild

hypoxia is a very physiologically relevant condition, given

that the oxygen concentration in most tissues is lower than

20%, which is the atmospheric oxygen concentration

(normoxia). Given that neural stem and progenitor cellshave been reported to show increased prolieration and

sel-renewal under mild hypoxic conditions8-10, studying the

eect o NSCs with respect to gaseous microenvironment

has the potential to advance research into the development

o NSC-based therapies or neurodegeneration.

We have successully developed the protocols to perorm

live cell imaging o rat NSCs using the CellASIC ONIX

Microuidic Platorm. By attempting various combinations

o these microenvironment parameters, we ound that the

rat neural stem cells exhibited dierent morphologies and

prolierated best under physiologic, mildly hypoxic

conditions, as reported in the literature8-10. In addition,

through the combination o high seeding density at

1 x 106/mL, polyornithine/laminin coating, continuous

perusion at 5 L/hour, and 3% oxygen (mild hypoxia) gas

microenvironment, the rat NSCs ormed a 3 mm x 3 mm x

0.1 mm neurosphere in 96 hours.

We also investigated the eect o varying cell density on

NSC dierentiation. The importance o cell density on the

dierentiation o NSCs has been recently established by

studies showing that NSC dierentiation can be regulated bycell-cell signaling, possibly through the epidermal growth

actor (EGF) and Notch signaling pathways11,12. Through live

cell imaging on a uorescent microscope, we discovered that,

while the peripheral cells around the neurosphere were

successully immunostained or two pluripotency markers,

nestin and Sox2, the image o the neurosphere itsel showed

condensed bright spots o Sox2 but no nestin, suggesting

that increased cell-cell contacts within the neurosphere may

aect NSC dierentiation.

In summary, we have demonstrated the combinatorial eect

o microenvironment parameters on rat NSCs and thecapability o imaging them with uorescent microscopy or

urther analysis. The platorm promises to acilitate assay

development or NSCs and provides a better-controlled

in vitromodel system or neurogenesis and neural

development research.

Available romwww.millipore.com.FEATURED PRODUCTS


CellASIC ONIX Microuidic System Package includes CellASIC ONIX Microuidic System, Maniold, Accessory

Box, and CellASIC ONIX FG Sotware

EV262

CellASIC ONIX Microincubator Package or Temperature and Gas Control: Includes CellASIC ONIX

Microincubator Controller, Microincubator Maniold, and Accessory Box

MIC230

M04S Microuidic Switching Plate or Mammalian Cells (4 Chambers) M04S-03-5PK

Adult Rat Hippocampal Neural Stem Cell Kit SCR021

Adult Rat Hippocampal Neural Stem Cells SCR022

Rat Neural Stem Cell Expansion Medium SCM009

Fibroblast Growth Factor basic, human recombinant GF003
http://www.millipore.com/?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/EV262?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/MIC230?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/M04S-03-5PK?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/SCR021?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/SCR022?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/SCM009?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/GF003?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/GF003?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/SCM009?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/SCR022?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/SCR021?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/M04S-03-5PK?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/MIC230?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/EV262?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/M04S-03-5PK?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/MIC230?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/EV262?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/?cid=BIOS-C-EPDF-1007-1301-BS


18/32

IntroductionCellular dierentiation is a undamental process in

developmental biology. Progenitor cells must have

the ability to dierentiate into more specialized cell

types or the body to respond to inections, eliciting

an immune response. Murine CD4+ T cells can give

rise to a variety o eector T, or T helper, cell subsets

depending on the nature o the immune response, and

subsequently release a distinct subset o cytokines.

During T cell dierentiation, a group o T helper (Th)

cell subsets play a critical role in both the innate and

adaptive immune response repertoire, deending the

body against oreign pathogens.

There are fve main subsets o dierentiated CD4+ T

cells: Th1, Th2, Th17, Treg, and Th cells. Each subtype

expresses a signature set o cytokines or transcription

actors that directs the immune response and regulates

dierentiation (Figure 1, page 2). For example, Th1

CD4+ T cells are important or protecting against

intracellular bacteria, ungi, and viruses, as well as being

involved in some autoimmune responses. These cells

mediate immune responses to intracellular pathogens

by producing intereron gamma (IFN-), and through

the secretion o IFN-, Th1 cells activate macrophages,natural killer (NK) cells, and CD8+ T cells. Unlike the

Th1 response, Th2 CD4+ T cells are oten associated

with humoral responses during which high levels o

Assessing enriched murine CD4+T cells dierentiated towardseector T helper cell lineages

with the Scepter 2.0 handheldautomated cell counterMark Santos, Wenying Zhang and Matthew Hsu


pathogen-specifc immunoglobulins are generated

to neutralize oreign organisms. Th17 cells play an

important role in the induction and propagation o

autoimmunity. Th17 cells signature cytokine is IL-17,

and IL-17 expression has been associated with many

autoimmune diseases such as multiple sclerosis,

rheumatoid arthritis, psoriasis, inammatory bowel

disease (IBD), as well as in allergic responses.

As CD4+ T cells dierentiate towards a specifc eector

T cell lineage, the cells dramatically change in size, a

unique physical hallmark o this dierentiation process.

We hypothesized that using the Scepter cell counter torapidly assess size distributions o cellular populations

would provide a quick, simple method or tracking T cell

dierentiation.

The Scepter cell counter captures the ease o

automated instrumentation and accuracy o impedance-

based counting using the Coulter principle in an

aordable, handheld ormat. The instrumentation has

been collapsed into a device the size o a pipette, and

uses a combination o analog and digital hardware or

sensing, signal processing, data storage, and graphicaldisplay in the orm o a histogram. The histogram output

provides a quick snapshot o cell size and density.


19/32

Materials and MethodsEnriched CD4+ T cell isolation

Mouse spleens were harvested rom 8-10 week old

C57BL/6 mice (Charles River Laboratories) and prepared

according to the standard guidelines or obtaining single

cell suspensions (splenocyte harvesting). Homogenized

spleens were passed through a nylon mesh to ensure

single cell suspensions, ollowed by treatment with redblood cell lysing buer (Sigma, Catalogue No. R7757) to

remove red blood cells rom the culture. Cells were gently

resuspended in Hanks Balanced Salt Solution (HBSS;

Sigma, Catalogue No. H9394) and counted to determine

splenocyte recovery, and then applied to a CD4 isolation

column (R&D Systems, Catalogue No. MCD4C-1000) to

enrich or CD4+ T cells.

CD4+ T cell dierentiation towards specifc

eector T cell lineages

Mouse CD4+ splenocytes were dierentiated to

one o three distinct lineages through a process o

activation, expansion, and re-stimulation. The cells

were activated by culturing on anti-CD3 coated plates

with the single addition o a combination o growth

actors and antibodies in precise amounts to begin the

dierentiation process (EMD Millipore, Catalogue No.

FCIM025161 or Th1 cells; EMD Millipore, Catalogue No.

FCIM025162 or Th2 cells; EMD Millipore, Catalogue No.

FCIM025163 or Th17 cells). Cells were urther expanded

and then re-stimulated at days 4 and 6, respectively.

Ater day 6, the dierentiated Th cell cultures were

secreting their signature cytokines.

Scepter cell counting

The Scepter cell counter was used to count samples

ollowing the detailed on-screen instructions or each

step o the counting process. Briey, the user attaches

a 40 m sensor tip, depresses the plunger, submerges

the sensor into the sample, then releases the plunger

drawing 50 L o cell suspension into the sensor. The

Scepter detects each cell passing through the sensors

aperture, calculates cell concentration, and displays a

size-based histogram as a unction o cell diameter or

volume on its screen. Scepter 2.1 sotware was then

used to upload test fles rom the device and perorm

subsequent data analysis to determine cell sizes or

mouse CD4+ T cell (undierentiated) versus Th1, Th2, or

Th17 eector T cell cultures (dierentiated).

In this study, we demonstrate a method or driving

murine CD4+ T cell dierentiation towards Th1, Th2,

or Th17 cells in vitroand measuring cell size changes

beore and ater this process using the Scepter cell

counter. We have employed Scepter technology or cell

volume/size determination to investigate the relationship

between cell dierentiation and cell size changes. We

confrmed T cell dierentiation status by using a ow

cytometry assay to measure intracellular cytokine

production or Th1, Th2, and Th17 cell types. Although

the Scepter cell counter was intended primarily as a

cell counting device, here we demonstrate how this cell

counter can also unction as a reliable tool or other

diverse biological applications.

Figure 1.

Eector CD4+ T cell lineage commitment. CD4 + T cellscan give rise to many subtypes depending on the type orequired immune response. A complex process includingactivation o T cell receptor, along with a local cytokineenvironment, polarize CD4+ T-cells to a defned lineage o

mature cytokine-producing helper T-cells and Treg cells.

DC

IL-12R

TH1

T-bet

STAT4

STAT1

IL-2R

TRE G

Foxp3

STAT5

IL-21R

TFHTH

Bcl-6

STAT3

IL-4R

TH2

GATA3

STAT6

STAT5a

IL-23R

TH17

RORt

STAT3

IL-12

CD80

CD28

TCR

CD28CD85

pMHCII

IFN

IL-18

IL-2

IL-4

IL-33

IL-6

IFN

IL-2

LT

IL-21

IL-6

TGFIL-23

TGF

IL-2

IL-4

IL-5

IL-13

IL-25

IL-17

IL-17F

IL-22

IL-21

IL-21

IL-17

TGF

IL-10

IL-35

naive
http://www.millipore.com/catalogue/item/FCIM025161?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025162?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025163?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025163?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025162?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025161?cid=BIOS-C-EPDF-1007-1301-BS


20/32

Evaluation o Th1, Th2, and Th17 cell lineages

by ow cytometry

All ow cytometry assays were perormed using EMD

Millipores FlowCellect Mouse Th intracellular cytokine

kits (EMD Millipore, Catalogue No. FCIM025123 or Th1

cells; EMD Millipore, Catalogue No. FCIM025124 or Th2

cells; EMD Millipore, Catalogue No. FCIM025125 or Th17

cells). All reagents and detailed instructions are provided

in the user guides or each kit. Please reer to each kits

specifc instruction or more detailed inormation. All

sample acquisition and data analysis was perormed

using the guava easyCyte system (EMD Millipore,

Catalogue No. 0500-4008).

Comparison o Scepter cell analysis with an

automated cell viability image-based system

Using the Scepter cell counter, cell sizes o CD4+ T cells

(undierentiated) and Th1 CD4+ T cells (dierentiated)

were analyzed in triplicate assays (three independent

experiments, plus three samples per experiment) andstandard deviation values were determined. In parallel,

both sets o samples were analyzed using a more

expensive automated cell viability analyzer.

ResultsMouse CD4+ T cells were treated with established

lineage-specifc actors or six days to drive

dierentiation towards eector T cell-specifc lineages:

Th1, Th2, or Th17 cells. Using the EMD Millipore mouse

dierentiation tool kits as described previously, we were

able to successully achieve cell dierentiation in an invitrosystem, and these results are captured using the

Scepter device based on cell size discrimination. Cross

validation studies were also perormed by ow cytometry

using EMD Millipores mouse intracellular cytokine kits to

veriy and confrm our results.

Scepter analysis o CD4+ T cell dierentiation

towards the Th1 lineage

Beore induction, undierentiated CD4+ T cells were

measured using the Scepter producing a mean cell

diameter o 6.4 m (Figure 2, blue histogram). Ater

six days in culture under the inuence o Th1 lineage-

inducing reagents, such as lineage specifc cytokines and

growth actors, resulting Th1 cells were measured using

the Scepter cell counter. The mean cell diameter o

this dierentiated cell population was 10.4 m (Figure

2, red histogram). Figure 3 shows brightfeld microscopy

analysis o the same cells, confrming that their average

diameter had indeed increased.

Figure 2.

Accurate discrimination between mouse CD4+ T cells(undierentiated) and dierentiated Th1 cells based oncell size. Using a 40 m sensor, the Scepter cell counterenabled the discrimination o cell types based on size withhigh resolution. Mouse CD4+ T cells were measured beoreand ater dierentiation towards the Th1 cell lineage. Ater sixdays in culture both cell types were measured and recorded.As indicated by the Scepter histogram data, cells increased insize rom 6 to 10 m, on average.

Figure 3.

Microscopy images beore and ater T cell dierentiation:confrmation that cell size is accurately measured by theScepter cell counter. CD4+ T cells prior to treatment underdierentiation conditions exhibited cell diameters around5-6 m, as shown in (A). In (B), six days ater exposure tospecifc cytokines and growth actors to induce dierentiationtowards the Th1 cell lineage in vitro, cells appear much largerwhen visualized at the same magnifcation.

A. Day One (1): Activation B. Day Six (6): Re-stimulation

400

320

240

Count

160

80

03 6

Diameter (m)

9 12 15 18

CD4+ T cells (Undifferentiated)

Th1 CD4+ T cells (Differentiated)
http://www.millipore.com/catalogue/item/FCIM025123?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025124?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025125?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/0500-4008?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025125?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025124?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025123?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/0500-4008?cid=BIOS-C-EPDF-1007-1301-BS


21/32 2

Figure 4.

Flow cytometry validates the dierentiationtowards Th1 cells, confrming the resultsobtained using the Scepter cell counter. Usingthe FlowCellect Mouse Th1 Intracellular CytokineKit (Catalogue No. FCIM025123), percentages odierentiated versus undierentiated CD4+ T cellswere determined. Undierentiated cells did notshow any signifcant expression o the signaturecytokine IFN-as seen in (A), however, ater sixdays in culture under lineage specifc conditionstoward Th1 cells, cells showed positive expressiono IFN-as seen in (B).

Validation o Th1 cell dierentiation

using ow cytometry

In order to validate that the cell types measured were

truly dierentiated cells, a ow cytometry assay was

perormed to measure intracellular cytokine production.

As shown in Figure 4, 91.2% o the day 6-stimulated

cell population expressed IFN-, a characteristic Th1

cell marker, confrming that the increase in cell size was

indeed a phenotype o dierentiated Th1 cells as studiedin Figures 2 and 3 (page 3).

The Scepter cell counter precisely measured cell

diameter changes across multiple samples (Table 1,

below). Moreover, the data showed that the Scepter

cell counter could achieve similar or more precise

measurements when compared to an alternative

benchtop instrument.

Table 1.

Scepter cell counter versus the automated cell viability image-based system: measuring cell size diameter or both naive CD4+ T cells (undierentiated)and Th1 CD4+ T cells (dierentiated).

Experiment #1 Experiment #2 Experiment #3

n=1 n=2 n=3 n=1 n=2 n=3 n=1 n=2 n=3

Average

diameter

(m)

Standard

Deviation

Scepter Values

Day 1

(Undierentiated)

6.33 6.36 6.4 6.14 6.24 6.66 6.33 6.34 6.43 6.36 0.14

Day 6

(Dierentiated)

11.0 11.02 11.07 10.05 10.05 10.28 9.97 9.97 9.98 10.38 0.50

Automated Cell Viability image-based System

Day 1

(Undierentiated)

7.01 6.48 6.87 6.49 6.57 6.91 6.41 6.88 6.92 6.73 0.23

Day 6

(Dierentiated)

11.31 11.40 11.88 10.25 10.37 10.79 10.02 10.13 10.28 10.71 0.66

104

103

102

101

100

100 101 102

CD4 (RED-HLog)

IFN(

YEL-HLog)

103 104

104

103

102

101

100

100 101 102

CD4 (RED-HLog)

IFN(

YEL-HLog)

103 104

A. Day One (1): Activation

B. Day Six (6): Re-stimulation
http://www.millipore.com/catalogue/item/FCIM025123?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025123?cid=BIOS-C-EPDF-1007-1301-BS


22/32

400

320

240Count

160

80

0

3 6Diameter (m)9 12 15 18


Th2 CD4+ T cells (Differentiated)

450

350

250Count

150

50

03 6

Diameter (m)

9 12 15 18


Th17 CD4+ T cells (Dif ferentiated)

Figure 6.

Flow cytometry confrmation odierentiation o enriched CD4+ T cellsto Th2 and Th17 cells. Cell types which

produced larger cell diameters as detectedby the Scepter cell counter were the samecells cultured or six days as shown in (B) and(D), whereas their progenitor cell types areshown in (A) and (C). Only cells in (B) and (D)expressed the signature cytokines IL-4 andIL-17 or Th2 and Th17 cells, respectively.

Figure 5.

CD4+ T cell dierentiationtoward Th2 and Th17cell lineages can beclearly identifed usingScepter based on cellsize. Using a 40 m sensor,the Scepter cell counterenables the discriminationo cell types based on sizewith high resolution. Thesize distributions o Th2and Th17 cell populationswere compared to the sizedistribution o the CD4+ Tcell progenitor cell type. BothTh2 and Th17 cells graduallyincreased in size rom 6to 10 m ater six days odierentiation.

CD4+ T cell dierentiation towards

the Th2 and Th17 lineages

CD4+ T cells were induced to dierentiate towards

both Th2 and Th17 cell lineages using EMD Millipores

Mouse Th Dierentiation Tool kits or Th2 and Th17

cells (Catalogue No. FCIM025162 and FCIM025163,

respectively). Beore induction, undierentiated CD4+ T

cells were measured using the Scepter cell counter as

having a mean cell diameter o 6.3 m (Figure 5, blue

histogram). Ater six days in culture under the inuence

o lineage-specifc reagents, cells were dierentiated

towards the Th2 and Th17 cell types. The resulting cell

populations were then measured using the Scepter cell

counter, which reported mean cell diameters o 10.1 m

and 9.8 m or Th2 and Th17 cells, respectively (Figure 5,

red histogram).

In order to validate that the cell types measured were

truly dierentiated cells and specifc or each cell

type described, we used ow cytometry to measure

intracellular cytokine production or the particularcell type in question. As shown in Figure 6, increased

expression o IL-4 or Th2 cells and increased

expression o IL-17 or Th17 cells confrmed that the

observed increases in cell size were true phenotypes o

dierentiated cells.

As with Th1 cell dierentiation, the Scepter cell counter

precisely measured cell diameter changes across multiple

samples o progenitor T cells and those dierentiated

toward Th2 and Th17 lineages (Table 2). Again, our data

showed that the Scepter cell counter could achievemore precise measurements when compared to an

alternative benchtop instrument.

A. Undierentiated CD4+ T cells versus Th2 cells

B. Undierentiated CD4+ T cells versus Th17 cells

104

103

102

101

100

100 101 102

CD4 (RED-HLog)

IL-4

(YEL-HLog)

103 104

104

103

102

101

100

100 101 102

CD4 (RED-HLog)

IL-4

(YEL-HLog)

103 104

104

103

102

101

100

100 101 102

CD4 (RED-HLog)

IL-17

(YEL-HLog)

103 104

104

103

102

101

100

100 101 102

CD4 (RED-HLog)

IL-17

(YEL-HLog)

103 104
http://www.millipore.com/catalogue/item/FCIM025162?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025163?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025163?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025162?cid=BIOS-C-EPDF-1007-1301-BS


23/32 2

Table 2.

Scepter cell counter versus the automated cell viability image-based system: Measuring cell size diameter or naive CD4+T cells (undierentiated), Th2 and Th17 CD4+ T cells (dierentiated). Data points with smaller standard deviations clearlydemonstrated the precision o the Scepter cell counter when compared to a more expensive cell counting instrument.

Table 1 (page 4) and Table 2 (below) were summarized in a bar graph showing the clear phenotypic dierence between

undierentiated and dierentiated CD4+ T cell populations, underscoring the utility o the Scepter cell counter to

provide a rapid, simple, accurate method or tracking immune response.

Th2 cell lineage

Th17 cell lineage


n=1 n=2 n=3 n=1 n=2 n=3 n=1 n=2 n=3

Average

diameter

(m)

Standard

Deviation

Scepter Values

Day 1

(Undierentiated)

6.24 6.26 6.30 6.37 6.37 6.38 6.40 6.42 6.43 6.35 0.07

Day 6

(Dierentiated)

10.07 10.11 10.24 9.92 10.05 10.11 9.88 9.97 10.27 10.07 0.13


Day 1

(Undierentiated)

7.00 7.18 7.20 7.01 7.15 7.33 7.35 7.47 7.59 7.25 0.20

Day 6

(Dierentiated)

10.68 10.95 10.99 11.04 11.10 11.48 10.57 10.98 11.36 11.02 0.29


n=1 n=2 n=3 n=1 n=2 n=3 n=1 n=2 n=3

Average

diameter

(m)

Standard

Deviation

Scepter Values

Day 1

(Undierentiated)

6.24 6.26 6.30 6.37 6.37 6.38 6.40 6.42 6.43 6.35 0.07

Day 6

(Dierentiated)

9.69 9.83 9.83 9.73 9.81 9.96 9.74 9.81 9.84 9.80 0.08


Day 1

(Undierentiated)

7.00 7.18 7.20 7.01 7.15 7.33 7.35 7.47 7.59 7.25 0.20

Day 6

(Dierentiated)

10.20 10.51 10.69 10.54 10.72 10.82 10.35 10.50 10.87 10.58 0.22


24/32

ConclusionsT cell dierentiation and its role in the immune response

are integral or both innate and adaptive immunity. The

importance o eector T cells and their ability to combat

against various types o pathogens, as well as maintaining

immune homeostasis, should never be underestimated.

In order to stay healthy, the body specifcally recruits CD4

eector T cells to execute its immune unctions. Dysregulated

Th cell unction oten leads to inefcient clearance o

pathogens, which can cause diseases o inammation and

impact autoimmunity.

Dierentiation o CD4+ T cells into ully unctional eector

T cells is characterized by both the production o signature

cytokines as well as a concomitant increase in cell size.

As demonstrated in this study, Th1, Th2, and Th17 cells

expanded rom approximately 6 to 10 m when compared

to the progenitor CD4+ T cell type. This expansion upon

dierentiation was clearly, accurately, and precisely measured

using the Scepter cell counter.

The Scepter provides a rapid, easy, and inexpensive method

or assessing naive CD4+ T cell dierentiation toward specifc

eector T cell lineages. This handheld, automated cell counter

delivers precise, reliable cell size measurements, which

can provide the researcher with a quick snapshot o the

dierentiation status and, ultimately, immune response.

Figure 7.

Cell diameter change during CD4+ T cell dierentiation(toward Th1 / Th2 / Th17 cell lineages) as measured by theScepter cell counter.

Reerences1. Wan, Y.Y., et. al. How diverse CD4 eector T cells and theirunctions. J Mol Cell Biol. 2009; 1(1): 2036.

2. Zhu, J., et. al. Dierentiation o eector CD4 T cell

populations. Annu Rev Immunol.2010; 28:445-89.

3. Zhou, L., et. al. Plasticity o CD4+ T cell lineage

dierentiation. Immunity 2009; 30(5):646-55.

4. Fietta, P., et. al. The eector T helper cell triade. Riv

Biol.2009;102(1):61-74.

Mouse CD4+ T cell Diameter (m)

0CD4+ T cells Th1

Differentiated(Day 6)

Undifferentiated(Day 1)

Th2 Th17

2

4

6

8

10

12


25/32 2

Reagents/Kits

Description Qty/Pk Catalogue No.

Scepter 2.0 Handheld Automated Cell Counter

with 40 m Scepter Sensors (50 Pack) 1 PHCC20040

with 60 m Scepter Sensors (50 Pack) 1 PHCC20060

Includes:Scepter Cell Counter 1

Downloadable Scepter Sotware 1

O-Rings 2

Scepter Test Beads 1 PHCCBEADS

Scepter USB Cable 1 PHCCCABLE

Scepter Sensors, 60 m 50 PHCC60050

500 PHCC60500

Scepter Sensors, 40 m 50 PHCC40050

500 PHCC40500

Universal Power Adapters 1 PHCCP0WER

Scepter O-Ring Kit, includes 2 O-rings and 1 flter cover 1 PHCC0CLIPGuava easyCyte 8HT Flow Cytometer 1 055-4008


FlowCellect Mouse Th1 Dierentiation Tool Kit FCIM025161



FlowCellect Mouse Th1 Intracellular Cytokine Kit FCIM025123




FEATURED PRODUCTS
http://www.millipore.com/catalogue/item/PHCC20040?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/PHCC20060?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/PHCCBEADS?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/PHCCCABLE?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/PHCC60050?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/PHCC60500?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/PHCC40050?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/PHCC40500?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/PHCCP0WER?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/PHCC0CLIP?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/055-4008?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025161?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025162?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025163?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025123?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025124?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025125?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025125?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025124?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025123?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025163?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025162?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/FCIM025161?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/055-4008?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/PHCC0CLIP?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/PHCCP0WER?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/PHCC40500?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/PHCC40050?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/PHCC60500?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/PHCC60050?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/PHCCCABLE?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/PHCCBEADS?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/PHCC20060?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/catalogue/item/PHCC20040?cid=BIOS-C-EPDF-1007-1301-BShttp://www.millipore.com/?cid=BIOS-C-EPDF-1007-1301-BS


26/32

Product Review

Autophagy:Mechanisms and connections

to apoptosisChandra Mohan, Ph.D., EMD Millipore Corporation

Phagophore

LC3

LC3Autophagosome Autolysosome

AutophagyInduction

Lysosome

Low NutrientsExerciseAdiponectinGhrelin

LKB1

CAMKK2

GrowthFactors

Ulk1/2

ProteinAggregate

ProteinAggregate

Ulk1/2

FIP200

Agt13

mTORmTORC1

AKT

AMPK

P

P P

P

P P

Autophagy is a highly regulated homeostatic

degradative process in which cells destroy and recycle

their own components via the lysosomal machinery.

In mammalian cells, autophagy is believed to occur

constitutively at basal rates. Under conditions o

extreme starvation, cells use this process to reallocatenutrients rom less important processes to more

essential processes required or survival. However, i

cellular damage becomes irreparable, cells can destroy

themselves completely by autophagy. Several processes

can be classiied under the general term autophagy.

Figure 1.

Steps o macroautophagy include induction, phagophoreormation, autophagosome maturation and autophagosome-

lysosome usion. LC3 is a small protein that is targeted tothe phagophore membrane by a ubiquitin ligase-like enzymecascade. LC3 mediates phagophore elongation to orm themature autophagosome.

However, one common element o autophagy pathways

involves the importation o cytoplasmic components

into the lysosome. In eukaryotes, autophagy unctions

solely as a degradative and remodeling pathway, while

in yeasts, autophagy also plays a role in biosynthesis.


27/32 2

Three types o autophagyGenerally, three types o autophagy have been

recognized. They are chaperone-mediated autophagy,

microautophagy, and macroautophagy. Chaperone-

mediated autophagy involves the direct translocation

o cytosolic proteins across the lysosomal membrane.

It requires cytosolic and lysosomal chaperones to

unold substrates. During microautophagy, cytoplasmis sequestered directly at the lysosomal surace by

separation and/or invagination o the lysosomal

membrane. In macroautophagy the sequestering o

membrane is distinct rom lysosome and it involves the

ormation o autophagosome that uses with lysosome,

which provides the hydrolytic enzyme machinery.

The used structure is termed as autophagolysosome.

The maturation o autophagolysosome requires

acidiication by H1-ATPase. Hence, several inhibitors

o H1-ATPase, such as Bailomycin A1, are shown

to diminish autophagy. Following their breakdown

macromolecules are released back into the cytoplasm

or reuse in the metabolic processes.

Steps o macroautophagyThe process o autophagy can be divided into several

phases. In the initial phase, the cell senses signals

released in response to lack o nutrients, hypoxia,

hormones or other sources o cell stress. These signals

can induce macroautophagy, either via inhibition

o mTOR or by AMPK activation o the ULK1/2kinase complex (Atg1 in yeast). mTOR is essential to

nutrient-sensing signal transduction and regulation o

cellutions 2012v4

Documents