Functional assays - Principles and Methods

J Paul Robinson

Pur

due

Uni

vers

ity C

ytom

etry

Lab

orat

orie

s

These slides are on the PURDUE CYTOMETRY WEB SITEThese slides are on the PURDUE CYTOMETRY WEB SITE

http://flowcyt.cyto.purdue.edu

These slides are on the PURDUE CYTOMETRY WEB SITEThese slides are on the PURDUE CYTOMETRY WEB SITE

http://flowcyt.cyto.purdue.edu

Presented at the Polish Society for Cytometry Meeting, Gdansk, Poland, October 18, 1998

Poland taken from Comsat C1 satellite

October 15, 1998 - you can clearly see Warsaw in the center. Top center-left is Gdansk.

Warsaw

Gdansk

Krakow

Gdansk

The goals of this presentation are:

• To identify the nature of functional assays in Cytometry

• To expand on how they operate

• To discuss the advantages and disadvantages of each

• To discuss the application of these assays

KineticsPrinciple of Time Measurements

• Live cells can be measured as easily as dead cells

• You only need a small number of cells in a changing environment

• End point assays can describe the activity of the cell

Cellular Functions

• Cell Viability

• Phagocytosis

• Organelle Function– mitochondria, ER

– endosomes, Golgi

• Oxidative Reactions– Superoxide

– Hydrogen Peroxide

– Nitric Oxide

– Glutathione levels

• Ionic Flux Determinations–Calcium

–Intracellular pH

• Membrane Potential

• Membrane Polarization

• Lipid Peroxidation

What do we measure?

TIME

Flu

ores

cenc

e

Fluorescent IndicatorsHow the assays work:• Superoxide: Utilizes hydroethidine the sodium borohydride reduced

derivative of EB

• Hydrogen Peroxide: DCFH-DA is freely permeable and enters the cell where cellular esterases hydrolyze the acetate moieties making a polar structure which remain in the cell. Oxidants (H2O2) oxidize the DCFH to fluorescent DCF

• Glutathione: In human samples measured using 40 M monobromobimane which combines with GSH by means of glutathione-S-transferase. This reaction occurs within 10 minutes reaction time.

• Nitric Oxide: DCFH-DA can also be used as an indicator for nitric oxide in a manner similar to H2O2

Organelle Function

• Mitochondria Rhodamine 123

• Endosomes Ceramides

• Golgi BODIPY-Ceramide

• Endoplasmic Reticulum DiOC6(3)

Carbocyanine

DCFH-DA DCFH DCFDCF

COOHH

Cl

O

O-C-CH3

O

CH3-C-O

Cl

O

COOHH

Cl

OHHO

Cl

O

COOHH

Cl

OHO

Cl

O

Fluorescent

Hydrolysis

Oxidation

2’,7’-dichlorofluorescin

2’,7’-dichlorofluorescin diacetate

2’,7’-dichlorofluoresceinCellular Esterases

H2O2

DCFH-DA

DCFH-DADCFH-DA

DCFHDCFH

DCF

H OH O 2 22 2

Lymphocytes

Monocytes

Neutrophils

log FITC Fluorescence.1 1000 100 10 1

0

20

40

60

cou

nts

PMA-stimulated PMNControl

80

HydroethidineHE EB

NCH2CH3

NH2H2N

H Br-NCH2CH3

NH2H2N

+

O2-

Phagocytic Vacuole

SODH2O2

NADPH

NADP

O2

NADPH Oxidase

OH-

O2-

DCFDCF

HE

OO22--

HH22OO22

DCFDCF

Example: Neutrophil Oxidative Burst

Both these images are cells stained to measure for H2O2 production.

Chondrocytes Neutrophil

Endothelial Adhesion Molecules

Neutrophil Counter Ligand

P-selectin (CD62P) s-Lex (CD15s)

E-selectin (CD62E) s-Lex, CD66, L-selectin, 2 integrins

Neutrophil Adhesion Molecules

Endothelial Counter Ligand

L-selectin (CD62L) s-Lex (CD15s)

CD11a/CD18

ICAM-1 (CD54), [iC3b, fibrinogen, factor X]CD11b/CD18

ICAM-1 (CD54), ICAM-2 (CD102)

CD11c/CD18 ?, [iC3b, fibrinogen]

Some examples of rapidly changing antigen expression systems

BACTERIALINFECTION

TNFIL-1

a

b cd

Endothelial Cells

E-selectin & P-selectin

ICAM-1

L-selectin &CHO ligands(e.g. sLex)

CD11b

Neutrophils

The circulating neutrophil (a) and the initiation of rolling (b) as molecular tethers are formed between selectin and CHO ligands on neutrophils and endothelial cells. If an adequate number of tethers are formed, the neutrophil completely decelerates and with chemotactic stimulation of the neutrophil, L-selectin is rapidly shed while other receptors like E-selectin, CD11b and ICAM-1 are up-regulated by cytokines and other inflammatory mediators (c). Firm neutrophil/endothelial cell adhesion is mediated by CD11b and ICAM-1 and is followed by emigration of the neutrophil through the endothelium (d).

E-selectin

ICAM-1

H2O2

O2-

OH-

NO.

NFB

TNF

VCAM

ROS

Bradykinin

NO.

? (NOO-)

-

+ +

+

++

Known and unknown interactions between neutrophils and endothelial cells. Nitric oxide (NO.) and reactive oxygen species (ROS) are produced by both neutrophils and endothelial cells thus the interaction between these reactive species becomes very complicated.

P-selectin

CD11b

membrane damage conjugated dienes

+ stimulatory effect- inhibitory effect

++

-

++

Oxidative Reactions

• Superoxide Hydroethidine

• Hydrogen Peroxide Dichlorofluorescein

• Glutathione levels Monobromobimane

• Nitric Oxide Dichlorofluorescein

Rat Pulmonary Artery Endothelial CellsOxidization via H2O2

Periodicity of Fluorescence

Purdue University Cytometry Laboratories

Meridian UltimaTM Analysis

Macrovascular Endothelial Cells in Culture

Time (minutes)0 60

Confocal System

Culture System

Step 1: Cell Culture

Step 2: Cell Wash

Lab-Tek

1 2

3 4

5 6

7 8

top view

side view

170 M coverslip

Step 3: Transfer to Lab-Tek plates

confocal microscopeoil immersionobjective

37o heated stage

stimulant/inhibitor added

Step 4: Addition of DCFH-DA, Indo-1, or HE

Hydrogen peroxide measurements with DCFH-DA

0

200

400

600

800

1000

1200

1400

1600

1800

2000

0 500 1000 1500 2000 2500 3000Time in seconds

cell 1

cell 2

cell 3

cell 4

cell 5

% c

hang

e (D

CF

fluo

resc

ence

)

525 nm

1 2

3

45

Step 6B: Export data from measured regions to Microsoft Excel

Step 7B: Export data from Excel data base to Delta Graph

Change in fluorescence was measured using Bio-Rad software and the data exported to a spread sheet for analysis.

Superoxide measured with hydroethidine

Export data from Excel data

base to Delta Graph

Export data from measuredregions to Microsoft Excel

cell 1

cell 2

cell 3cell 4

cell 5

Change in fluorescence was measured using Bio-Rad software and the data exported to a spread sheet for analysis.

%ch

ange

(D

CF

fluo

resc

ence

)

-200

0200

400600800

100012001400

16001800

cell 1

cell 2

cell 3

cell 4

cell 5

Time in seconds

1000 1200 1400 1600 1800600 800 200 400

Promyelocyte

NeutrophilMyelocyte

Metamyelocyte

Padma Narayanan figures hl60.ppt

0

6

12

18

24

30

36

0 HOURS 24 HOURS 48 HOURS 72 HOURS 96 HOURS

Ch

ange

in M

ean

DC

F F

luor

esce

nce

0 ng/ml PMA

8 ng/ml PMA

50 ng/ml PMA

a

ab

b1

c1

c

d

ed1

e1

0

6

12

18

24

30

36

0 0.1 1 5 10 20 50

Diphenyleneiodonium chloride [M]

0 HOURS PMA (8 ng/ml)

0 HOURS PMA (50 ng/ml)

96 HOURS PMA (8 ng/ml)

96 HOURS PMA (50 ng/ml)

Ch

ange

in M

ean

DC

F F

luor

esce

nce

DCF Fluorescence

EB Fluorescence

0

5

10

15

20

Ch

ange

in M

ean

Ch

ann

el F

luor

esce

nce

Passage 28 Passage 60

HL-60 cells

Phagocytosis• Uptake of Fluorescent labeled particles

• Determination of intracellular or extracellular state of particles

How the assay works:• Particles or cells are labeled with a fluorescent probe

• The cells and particles are mixed so phagocytosis takes place

• The cells are mixed with a fluorescent absorber to remove fluorescence from membrane bound particles

• The remaining fluorescence

represents internal particlesFITC-Labeled Bacteria

Trypan Blue

FITC-Labeled Bacteria

pH Sensitive Indicators

• SNARF-1 488 575

• BCECF 488 525/620

440/488 525[2’,7’-bis-(carboxyethyl)-5,6-carboxyfluorescein]

Probe Excitation Emission

Applications• Probe Ratioing

– Calcium Flux (Indo-1) – pH indicators (BCECF, SNARF)

Molecule-probe Excitation EmissionCalcium - Indo-1 351 nm 405, >460 nmCalcium- Fluo-3 488 nm 525 nmCalcium - Fura-2 363 nm >500 nmCalcium - Calcium Green 488 nm 515 nmMagnesium - Mag-Indo-1 351 nm 405, >460 nmPhospholipase A- Acyl Pyrene 351 nm 405, >460 nm

Ionic Flux Determinations• Calcium Indo-1

• Intracellular pH BCECF

How the assay works:

• Fluorescent probes such as Indo-1 are able to bind to calcium in a ratiometric manner

• The emission wavelength decreases as the probe binds available calcium

Time (Seconds)0 36 72 108 144 180

RAT

IO [s

hort

/long

]0

200

400

600

800

1000

StimulationStimulation0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 50 100 150 200

Rat

io: i

nten

sity

of 4

60nm

/ 40

5nm

sig

nals

Time (seconds)

Flow Cytometry Image Analysis

1

2

33

2

1

405/35 nm460 nm

Calcium ratios with Indo-1

Changes in the fluorescence were measured using the Bio-Rad calcium ratioing software. The same region in each wave length was measured and the relative change in each region was recorded and

exported to a spread sheet for analysis.. Export data from measured regions to Microsoft Excel Export data from Excel data base to Delta Graph

50 100 150 2000

0.1

0.20.3

0.40.5

0.60.7

0.8

0

cell 1 cell 2 cell 3

Ratio: intensity1 (460nm) / intensity2 (405/35nm)

Probes for Ions

• INDO-1 Ex350 Em405/480

• QUIN-2 Ex350 Em490

• Fluo-3 Ex488 Em525

• Fura -2 Ex330/360 Em510

Membrane Potential• Oxanol Probes • Cyanine Probes

How the assay works:• Carbocyanine dyes released into the surrounding media as cells depolarize

• Because flow cytometers measure the internal cell fluorescence, the kinetic changes can be recorded as the re-distribution occurs

Time (sec)

Gre

en F

luor

esce

nce

Repolarized Cells

051

210

24

0 300 1500 1200 2400

Time (sec)

051

210

24G

reen

Flu

ores

cenc

e

PMA Added fMLP Added

Depolarized Cells

Lipid Peroxidation

• Probe: 5 M cis-paranaric acid (Molecular Probes)

How the assay works:• Cis-paranaric acid is a naturally fluorescent fatty acid which has

4 conjugated double bonds which become targeted by lipid peroxidation reactions with a subsequent loss of fluorescence

Lipid Peroxidation Paranaric Acid

0 1

024

TIME (Seconds)

Over time - Paranaric acid loses its fluorescence as the double bonds are destroyed

TIME (Seconds)

Data on left taken from Hedley, et al, Cytometry, 13: 686-692, 1992

Cartoon of the curve that would be derived from the data at left.

“Caged” Photoactivatable Probes

• Ca++: Nitr-5

• Ca++ - buffering: Diazo-2

• IP3

• cAMP

• cGMP

• ATP

• ATP--S

Available Probes

Principle: Nitrophenyl blocking groups e.g. nitrophenyl ethyl ester undergoes photolysis upon exposure to UV light at 340-350 nm

Release of “Caged” Compounds

UV Beam

Release of “Cage”

Culture dish

UV excited

Control Region

Caged Nitric Oxide study

Export data from measuredregions to Microsoft Excel Export data from Excel data dbase to Delta Graph

Regions were selectively excited using UV light to release the cage nitric oxide. Images of the excited and adjacent control

region were then collected.

Time (seconds) after UV FLASH

0 20 40 60 80 100 120 140 160

0

50

100

150

200

250

Flu

ores

cenc

e E

mis

sion

at 5

15 n

m

0

FRAPIntense laser BeamBleaches Fluorescence

Recovery of fluorescence

10 seconds 30 secondsZero time

Time

%F

Conclusions & Summary

• Oxygen radicals

• Nitrogen radicals

• Antioxidants

• Cell viability

• Organelle function

Functional Studies In Cytometry

• Lipid peroxidation

• Membrane potential

• Calcium fluxes

• pH changes

Acknowledgements• Kathy Ragheb

• Gretchen Lawler

• Steve Kelley

• Monica Shively

• Dave Whittinghill

• Stephanie Sincock

• Karen Cornell

• Karin Kooreman

• Nian-Yu Li

Padma Narayanan (Smith Kline)

Wayne Carter (Pfizer)