Flow Cytometry Core

A Resource of the Section of Immunobiology,

Department of Internal Medicine, and the YCCC

Presentation Overview

• Flow cytometry background and technical review

• Yale Flow Core: history, capabilities, people, and operation.

• Use of flow cytometry to study memory B cells in my own lab.

What is Flow Cytometry?

• The analysis of single cells, particles, or other discrete elements as they flow past one or more focused light sources based on reflected, scattered, or fluorescent light generated by those light sources.

Flow CellFlow Cell

Injector Tip

FluorescenceFluorescencesignalssignals

Focused laserFocused laserbeambeam

Sheath fluid

Forward Angle Light ScatterForward Angle Light Scatter

FALS Sensor

Laser

90 Degree Light Scatter90 Degree Light Scatter

FALS Sensor

90LS Sensor

Laser

Laser

Fluorescence DetectorsFluorescence Detectors

Freq

Fluorescence

FALS Sensor

Fluorescence detector(PMT3, PMT4 etc.)

488 nm laser

+

Fluorescence Activated Fluorescence Activated Cell SortingCell Sorting

Charged Plates-Attract and focusdroplets of oppositecharge

Single droplets sortedinto test tubes

FALS Sensor

Fluorescence detector

Computer analysis of detector data- “gating”

leads to + or - charging of droplet

-

Optical Optical FiltersFiltersDichroic Filter/Mirror at

45 deg

Reflected light

Transmitted Light

Light Source

Standard Band Pass FiltersStandard Band Pass Filters

Transmitted LightWhite Light Source

630 nm BandPass Filter

620 -640 nm Light

Standard Long Pass Standard Long Pass FiltersFilters

Transmitted LightTransmitted LightLight SourceLight Source520 nm Long Pass Filter520 nm Long Pass Filter

>520 nm >520 nm LightLight

Transmitted LightTransmitted LightLight SourceLight Source575 nm Short Pass Filter575 nm Short Pass Filter

<575 nm <575 nm LightLight

Standard Short Pass FiltersStandard Short Pass Filters

PMT

PMT

PMT

PMT

DichroicFilters

BandpassFilters

Flow Cytometry OpticsFlow Cytometry Optics

Laser

1

2

3

4

Flow cell

What can flow be used for?

• Expression of cell surface or intracellular proteins or neo-epitopes such as phospho-proteins generated via cell signaling (after permeabilization) using fluorescently tagged Abs.

• Use of small molecule, protein, or particle-based probes to detect: Ca++ flux, pH, mitochondrial polarization (apoptosis surrogate), caspase activation, cell division via dye dilution (e.g. CFSE), phagocytosis, DNA content (cell cycle analysis) or proliferation (BrdU incorporation).

• Detection of intrinsic cell fluorescence based on expression of fluorescent proteins from reporter constructs.

• Analysis of cell size and complexity using light scatter.• Multiparameter analysis to determine cellular heterogeneity

and to link properties to cell phenotypes in complex mixtures.

• Quantitative technique to enumerate specific cell types.• Preparative method that can combine any of the above

techniques isolate cells or even subcellular fractions at a rate of up to 50K events/second.

The Yale Flow Facility

History• Began as 1 FACScan and FACStar plus, supported by HHMI and Section of Immunobiology.

• FACScalibur added in 94.• FACSVantage in 98.• FACS-DIVA upgrade in 2001-provided gratis by BD.• Winter 2002: Current core opened in TAC with space and instruments contributed by Internal Medicine and purchase of additional used MoFlo by YSM. Custom-built rooms.

• Initial configuration: 1 FACScan, 4 FACSCaliburs (3 from Int. Med), 2 sorters.

• Shared Instrument Grant for FACSAria in 2003.• Fall of 2004: Purchase of LSRII-with UV capability for DNA analysis.

• November 2004: YCCC merger completed.

Instrumentation• FACS Analysis:

– 3 Color analysis: FACScan– 4 Color analysis: 4 FACScaliburs– 11-Color analysis: LSRII (new)

• Sorters:FACSVantage DIVA: hi-speed digital stream in air sorter with UV, 488, 633; 4-way sort; cloning; aerosol containment.

MoFlo: hi-speed digital stream in air sorter with 488, 633; cloning

FACSAria: hi-speed digital cuvette sorter with 407, 488, 633; hi sensitivity, 13-color; 4-way sort; cloning; aerosol containment

TAC Building Room S617

31’

15’ 15’

7’-10” 7’-10”7’-10”

2’ 2’ 2’ 2’

2’ 2’

FA

CS

Aria

/Com

pute

r

8’-9

”W x

4’-5

”D

Desk4’-7”W x 2’-6”D

MoFlo3’ D x 6’ W

2’ W x 2’D

FACS Vantage 4’ D x 3’ W

3’ W x 3’ D

Fili

ng

Ca

bin

et

2

’ W x

5’ D

2’-6

”

10’ 10’4’-7”

2’-6

”

Scale 0.3”-1’ - Bench Space

Lasers and ColorsInstruments Laser Excitation Laser Line (nm) Fluorescence Channel FluorochromesBD FACScan Argon (L1) 488 FL1 Green FITC Alexa Fluor 488

FL2 Yellow PE PI EMAFL3 Red PE-Texas Red PE-Cy5 PerCP PerCP-Cy5.5 PE-Cy7

BD FACSCalibur Argon (L1) 488 FL1 Green FITC Alexa Fluor 488FL2 Yellow PEFL3 Red PE-Texas Red PE-Cy5 PerCP PerCP-Cy5.5 PE-Cy7

Red Diode (L2) 635 FL4 Red APC Alexa Fluor 647BD FACSVantage Argon (L1) 488 FL1 Green FITC Alexa Fluor 488

FL2 Yellow PE PI EMAFL3 Red PE-Texas Red PE-Cy5 PerCP PerCP-Cy5.5 PE-Cy7FL6 UV Hoechst Alexa 350 Indo-1

HeNe (L2) 633 FL4 Red APC Alexa Fluor 647FL5 InfraRed APC-Cy7

BD FACSAria Argon (L1) 488 Green FITC Alexa Fluor 488Yellow PERed PE-Texas Red PE-Cy5 PerCP PI EMAFar Red PerCP-Cy5.5Infra Red PE-Cy7

HeNe (L2) 633 Red APC Alexa Fluor 647Infra Red APC-Cy7

Violet (L3) 407 Infra Red Alexa Fluor 405 Pacific BlueBD LSR II Argon (L1) 488 Green FITC Alexa Fluor 488

Yellow PERed PE-Texas Red PE-Cy5 PerCP PI EMAFar Red PerCP-Cy5.5Infra Red PE-Cy7

HeNe (L2) 633 FL4 Red APC Alexa Fluor 647Infra Red APC-Cy7

UV (L3) 355 VioletBlue Alexa Fluor 405 Pacific Blue

Violet (L4) 407 Blue Alexa Fluor 405 Pacific BlueCytomation MoFlo Argon (L1) 488 Green FITC Alexa Fluor 488

Yellow PE PI EMARed PE-Texas Red PE-Cy5 PerCPFar Red PerCP-Cy5.5Infra Red PE-Cy7

HeNe (L2) 633 FL4 Red APC Alexa Fluor 647Infra Red APC-Cy7

Capabilities and Techniques

• User operated 3, 4, and 11-color analysis.• Technician-assisted analysis on request for an additional fee.

• Multicolor hi-speed digital sorting:– 4-way– Cloning/single cell or multicell– Sterile– UV, 407, 488, and 633 laser lines– Operator performs most sorts; user-operation an option for experienced FACS Aria users.

• Techniques: cell surface markers, cytokines, intracellular staining, live-dead discrimination, Ca++-signaling, DNA/cell cycle analysis, FRET, subcellular fractions, detection of almost any fluorescent molecule.

• Assistance with data analysis: two workstations with appropriate software.

Personnel• Mark Shlomchik has run facility for the last 7 yrs. Currently my 5% effort is supported by a PPG on which I am one of the PIs.

• Two sorter operators paid by effectively 50% by HHMI: Tom Taylor and Gouzel Tokmoulina.

• One R+D tech who supervises FACS Aria and analyzers: Geoff Lyon.

• One new R+D tech who oversees the analyzers, billing and training: Don Foster.

User Support and Resources• Consultation on sorts, analysis, etc.

before, during or after the experiment, as needed, no charge.

• Regular training sessions for all instruments.

• Broadcast announcements via email about new policies, unexpected downtime, etc.

• Sponsorship of training seminars on data analysis, new reagents, techniques.

• Negotiated discounts with a variety of vendors.

User Support and Resources• New web site: designed and

maintained by Gouzel Tokmoulina.– Description of equipment– Facility rules– Sign-up– Resources and information

• Web-based scheduling for all analyzers.

Usage Statistics

• Analysis: ~950 hr./month• Sorting: ~325 hr./month• 119 Active laboratories• >486 Registered individual users

Budget

• Sources of income– User fees: ~$440K– YCCC: $39K– HHMI: ~$112K

• Costs– Salaries: ~$300K/yr. Salary+fringe for 4 people and administrative support.

– Maintenance contracts: $135K/yr. (some deferred due to prepaid contracts.

– Other: ~$25K/yr.

Breakdown of Key Fees

• User-operated analysis: $14/hr.• User-operated LSRII or Aria (analysis only): $22/hr.

• Operator-performed sorting or analysis: $68/hr.

• Training: $30/hr. for up to three people (1 hr. sessions).

Future Plans• Additional LSRII in one to two years. We have applied for a Shared Instrument Grant that is pending review.

• Further application development and customer support and training.

• Expansion with a satellite facility in the Amistad Building to support Stem Cell, Vascular Biology and Human Immunology programs: will need a new sorter and an LSRII analyzer. All users could use either facility.

• Expanded education mission.• Ideas for what you want or need?

Applications Examples-Study of B cell Memory• Identifying memory cells and phenotyping them using BrdU labeling.

• Sorting memory cells for mRNA or subsequent functional analysis.

• Using FACS to confirm the microarray data.

Definition of a “Memory Cell”

• A cell that has previously responded to antigen and that persists in a resting state for a long period of time after initial exposure.

• What are the features that distinguish resting “memory” and naïve B cells?

Eswitch

VDJ C M

s

Balb/c IgMa, no secreted exonBl/6, VH186.2

Signals for production of secreted IgM have been deleted.Only membrane-bound IgMa is produced by the transgene.

Pairing of the VH186.2 variable region gene withendogenous 1 light chain produces antibody specific forthe hapten NP.

This provides a system in which the functions of B cellsand antibody can be distinguished.

Membrane IgM (mIgM)Transgene Construct

Ig Transgenic mice with an increased frequency of

hapten NIP-specific B cells

Expansion of Antigen-specific Population 12

weeks after Immunization

Summary of NP+ Expansion after Immunization

BrdU is administered intraperitoneally every 12 hours

on the days indicated above.

BrdU Labeling Strategy

BrdU-positive Ag-specific B cells present in mIg and (m+s)Ig mice

12 weeks post immunization

n=5-14 mice

BrdU+NP+ Half life >8wks

Decay Kinetics of BrdU-labeled Memory B cells is Equivalent in mIg and

(m+s)Ig mice

BrdU-positive, NP+ B cells present in mIg and (m+s)Ig

Immune mice were compared with NP+ cells in Alum control mice

Resting Memory Cells Have High CD80 Expression

Bold=immune

A System for Generating Large Numbers of Memory Cells for

Further Study: Hyperimmunization of mIg mice

NP-CGG AssayNP-CGG

d0- 4wk 8 12 20wk16

A.

1.66 7.96

NP+

B22

0+

Naive Two Doses

12 weeks post 2nd immunization

B.

C.

Naïve or Immunized -NP Splenocytes

FACS Sorting

RNA Isolation

Affymetrix Hybridization

APPROACH

Statistical Analysis

Data Mining

1.68

93.8

Kappa

NIP

3.16

92.4

Naive

Memory

Biological Replicates

Naïve NPMemory NPGC NP

#134

Labeled cRNA Preparation

#2446

PI

97.3

PE-anti-

AA4.1

35.1

# Cells 50.8

Side

Scatter

FACS Isolation of NP+ Splenic B Cells for Affymetrix and qPCR

Analysis

Fitc-anti-B220 APC-NIP

Naive

Memory

77.9

Bonnie JhD-/- Jk-/- mice

Bonnie JhD-/- mice 12 weeks post 2nd immunization

4oC; NaAzide 0.05%; FCS

17.4

79.3

Biot-anti-KappaAPC-NIP

51.7

Fitc-anti-B220

# Cells97.9

PI

Side

Scatter

Cell Surface Molecules: M>N

(Affy and qPCR ConfirmedMela (80-kD Melanoma Antigen)

Emp1 (epithelial membrane protein 1)

Bmpr1a (bone morphogenetic protein receptor, type 1A)

Atp11a (ATPase, class VI, type 11A)

Myadm (myeloid-associated differentiation marker)

Adora2a (adenosine A2a receptor)

CD80

CD36

Acknowledgements

• Tom, Geoff, Gouzel, and Don• HHMI, Internal Medicine, YCCC• YSM Administration• Shannon Anderson and Mary Tomayko

• Today’s speakers