Flow cytometry workshopBD Accuri™ C6

October 2015

Single cells in suspension pass a laser beam

The cells produce characteristical light signals depending on cell type and preparation that will be analyzed by specific detectors

Priciple of flow cytometry

Algae

Bloodcells

DNA/RNA

Bacteria

What can be analyzed?

• Its relative size (Forward Scatter - FSC)

• Its relative granularity or internal complexity (Side Scatter - SSC)

• Its relative fluorescence intensity

Which parameters can be analyzed?

Forward Scatter—diffracted light Related to cell surface area Detected along axis of incident light in the forward direction

Side Scatter—reflected and refracted light Related to cell granularity and complexity Detected at 90° to the laser beam

Right Angle Light Detector

Cell Complexity

Forward Light Detector Cell Surface Area

Incident Light

Source

Properties of FSC and SSC

Basophil Granulozyte Lymphozyte

Eosinophil Granulozyte

Neutrophil Granulozyte

Monozyte

Thrombozytes

Erythrozytes

Granulocytes

Lymphozytes

(FSC)

Monozytes

Debris(S

SC)

Human whole blood

Lysed whole blood

The fluorochrome absorbs energy from the laser. The fluorochrome releases the absorbed energy by:

vibration and heat dissipation. emission of photons of a longer wavelength.

= 488 nm

Emitted Fluorescent Light Energy

Antibody

IncidentLight Energy

FluoresceinMolecule

520 nmHO

CO2H

O

C

What is fluorescent light?

Red laser 635 nmBlue laser 488 nm

FITC

488 nm 635 nm

PerCP

PE

APC

Excitation spectra

Blue laser

Red laser

Emission spectra

Relative fluorescence intensity

Num

ber o

f eve

nts

FITC

100 101 102 103 104FITC

FITC

FITC

FITC

FITC

FITC

FITC

FITC

FITC

Emitted fluorescence intensity proportional to binding sites

Fluorescence intensity

|480

|580

|560

|500

|520

|540

|620

|600Wave length(nm)

FITC

PE

530/30 585/42 488nm

100-

80-

60-

40-

20-

0-

Rel

ativ

e in

tens

ityFluorescence spillover

Increase of percentage

Compensation

The BD Accuri™ C6 Flow Cytometer

An affordable, full-featured, easy-to-use flow cytometer.Two lasers and six detectors

Components of a flow cytometer

• Fluidic system

• Optical system (excitation and detection)

• Electronic system and software

Fluidics

• Non-pressurized, peristaltic pump-driven system

• Patented pulse dampeners• User adjustable flow rate and

core diameter for a variety of applications

• Volume measurement for absolute counts

• Minimum sample volume 50 µL• Up to 10,000 events/second

Fluidics: Enhanced sample handling

• Microprocessor-controlled peristaltic pumps enable direct volume measurement

• Many types of sample tubes may be used• Falcon “FACS” tubes• Eppendorf• Micro-capillary tubes

• Add reagents or cells during a run• BD CSampler™ Accessory for automated

sample introduction

Fluidics: Sample Flow

Low pump speed High pump speed

Sample

LaminarFlow

Sheath SheathSample

High 66µl/min

LaminarFlow

Sheath Sheath

Low14µl/min

Excitation optics consist of: A laser Lenses to shape and focus the laser beam

Collection optics consist of: A collection lens to collect light emitted from the particle-

laser beam interaction A system of optical mirrors and filters to route specified

wavelengths of emitted light to designated optical detectors

Optics

460 500 540 460 500 540

LP 500 BP500/50

Longpass Bandpass

Optical filters

Signal amplification:• Photo diode• PMT (Photomultiplier Tube)

Optics: Detecors

Optics: System overview

FSC

SSC

488 nm solid state laser

640 nm diode laser

PMTs for fluorescence

Detection

Diodes for scatter detection

Compact optical system design reduces cost and

eliminates alignment issues

510/15540/20565/20610/20780/60

Instrumentconfiguration3 blue 1 red

User changeable

optical filters

BD Accuri™ C6 default optics

DetectorFL1

FL2

FL3

FL4

Filter533/30nm

585/40nm

670nm LP

675/25nm

FluorochromeFITC

PE

PerCP, PerCP-Cy5.5

APC

ColorGreen

Yellow/Orange

Dark Red

Red

“3 blue 1 red”

8-Peak Validation Beads (FL1, FL2, FL3)

6-Peak Validation

Beads (FL4)

FL1 = 533/30 FL2 = 585/40 FL3 = 670LP

101 102 103 104 105 106 101 102 103 104 105 106 101 102 103 104 105 106

FL4 = 675/25

101 102 103 104 105 106

Detectors – balanced at manufacture

39,27139,271Event 1Event 2Event 3

FSC SSC FITC PE0 60 120 90

10 160 65

30 650 160

FCS data

185Time

19002,688

31003,189

PE-A

1900

90

185 3100

PE-A

FITC

-A

1900

90

FITC

-A185 3100

Electronics: the result

Pre-optimized detectors – adjust view

Lymphocytes

Zoom Area

Zoom OutZoom Again

Optics: Advantages of pre-optimized voltage

• Greatly reduces risk of lost data due to improper set-up

• No specialist training or dedicated operator required

• Predictable, reproducible analysis relative to sample type and application

• Saves time and sample

• Attenuation filters (for too-bright signals) give controlled signal reduction

• Predictable fluorescence spillover

Compensation depends on

• Instrument configuration (filter and lasers)

• Used colors in panel

• Voltage (instrument settings)

predictable spillover for panels with same colors

Predictable spillover

FITC PE PerCP PerCPCy5.5 PE-Cy7 APC

FL1 (533 BP) ---- 3.20 0.00 0.00 0.50 0.00

FL2 (585 BP) 7.50 ---- 0.00 0.00 1.50 0.00

FL3 (670 BP) 1.00 19.50 ---- ---- ---- 0.80

FL4 (675 BP) 0.00 0.00 3.00 12.00 0.00 ----

Suggested Compensation Values for the BD Accuri C6

Compensation Controls – Beads

Advantages of compensation beads– Easy to use

– Do not require cells

29

FITC

PE

BD Accuri™ C6 Software

Sample Grid

CytometerStatus

Fluidics Controls

Run Criteria

Threshold and Compensation

Real Time Updates

Analysis andGating Tools

RegionsQuadrant Markers

HistogramsDot plotsDensity plots

Plot Statistics

Software: using Templates

BD Templates on the web

http://www.bdbiosciences.com/go/templates

Options in Flow CytometryApplications and staining targets

IntranuclearTranscription factors

Cell surfaceLineage markerActivation markerCytokine & chemokine receptors

IntracytoplasmaticCytokine, chemokines, various proteins

Apoptosis & cell viability

Proliferation

Quantification in supernatant, plasma, serum or cell lysates:Cytokines, Chemokines, immunoglobulins or signaling proteins

Supernatant, plasma, serum or cell lysates:ELISA, ELISPOT and Western Blot

Intracytoplasmatic or intranuclearCell Signaling Proteins

Microbiologywater analysis, bioprocessingMolecular biologytransfection

Human PBMC: T cell phenotyping

Lymphocyte Gate

Isotype-FITC CD45RA-FITC

CD

8-PE

-Cy7

CD3-APC

CD

4-PE

CD3-APC Isotype-FITC CD45RA-FITC

Gate:CD3+ CD8+

Gate:CD3+ CD4+

T cell phenotyping T regs: CD4-FITC FOXP3-APC

Sample B, C6

FOXP3-APC

CD

4-FI

TC

FOXP3-APC

CD

4-FI

TC

Sample A, C6

Methods: Blood samples were stained with CD4-FITC, fixed, permeabilized, and stained with FOXP3-APC. The background present in the CD4+FOXP3+ gate shown was 0% for the CD4+-FITC, unstained-APC control (data not shown).

2.4%

1.8%

Cell Cycle Analysis with Propidium Iodide

G1

S G2/M

Sub - G1

Camphthotecin induced murine thymus Annexin V

609.525 1.600.0001.000.000

0228.572

100.000

SSC-A

FSC-A

A01 Thymus campto unstainedGate: [No Gating]

P139,2% P2

37,0%

P139,2% P2

37,0%

101 107.2102 103 104 105 106

101

107.2

102

103

104

105

106

Annexin V APC - FL4-A

PI - FL2-A

A01 Thymus campto unstainedGate: P1 and P2

R10,1%

R266,2%

R30,1%

P30,2%

R10,1%

R266,2%

R30,1%

P30,2%

101 107.2102 103 104 105 106

101

107.2

102

103

104

105

106

Annexin V APC - FL4-A

PI - FL2-A

A02 Thymus Campto PIGate: P1 and P2

R10,1%

R239,7%

R312,9%

P320,9%

R10,1%

R239,7%

R312,9%

P320,9%

101 107.2102 103 104 105 106

101

107.2

102

103

104

105

106

Annexin V APC - FL4-A

PI - FL2-A

A03 Thymus Campto Annexin APCGate: P1 and P2

R162,8%

R219,6%

R30,3%

P30,0%

R162,8%

R219,6%

R30,3%

P30,0%

101 107.2102 103 104 105 106

101

107.2

102

103

104

105

106

Annexin V APC - FL4-A

PI - FL2-A

A04 Thymus Campto PI AnnexinGate: P1 and P2

R134,2%

R219,2%

R332,7%

P31,0%

R134,2%

R219,2%

R332,7%

P31,0%

Assessment of Cell ViabilityBD Horizon™ Fixable Viability Stain 520

• Amine-reactive dye useful for live/dead discrimination

• Compatible with fixation and permeabilization protocols

• Optimized all common laser lines:–Blue laser: - FVS520 (FITC channel) - FVS570 (PE channel)

–Red laser: - FVS660 (APC channel)

Examples of multicolor flow cytometric analysis of phosphorylated Stat3 expression by “viable” activated PBMCs.

Stat3 (pY705) AlexaFluor® 647

only living cells no live cell discrimination

BD Horizon™ Fixable Viability Stain 520

SSC

38

T cells only 0.4%

DC + T cells1 : 4 52.4%

In Vitro T cell proliferation: T cells + dendritic cells

39

CFSE

Gate = CD3+ Cells% of CD3+

T cellsProliferating

Courtesy of: Reddy P, Sung Y. Department of Pediatrics, University of Michigan, Ann Arbor, MI

FSC

-A

Flexibility in optical configuration - filters

Detector Filter options Fluorophore

FL1 530/30 BP (standard) FITC, GFP, YFP, CFSE, AF-488

515/15 GFP

FL2 585/40 BP (standard) PE, PI

540/20 BP YFP

FL3 670 LP (standard) PerCP, PerCP-Cy5.5, PE-Cy5, PE-Cy7, PI, 7-AAD

610/20 BP RFP, PI, PE-CF594

FL4 675/25 BP (standard) APC, PE-Cy5, AF-647

“3 blue 1 red”

Applications: GFP/YFP

Separation of GFP and YFP signals

Standard Filters GFP /YFP Combo

FL2:

585

/40

FL2:

540

/15

GFPYFP GFP

YFP

Detection of GFP expression in bacteria

Courtesy of: Tim F. Cooper, Dept. of Biology and Biological Chemistry, University of Houston, Houston,TX.

+ GFP

- GFP

E. coli B mixture

E. coli B+ GFP

plasmidE. coli B wild

type

FL1: 530 BP FL1: 530 BP FL1: 530 BP

Applications: Human Th1/Th2/Th17 CBA Kit

Optics: Selectable Lasers Module

FSC

SSC

510/15540/20565/20610/20780/60

3 blue 1 red2 blue 2 red

4 blue

User changeable optical filters

Selectable Lasers

Selectable Lasers Module provides flexibility

Reassign laser and detector associations

Standard: 488 FL1,2,3 640 FL42-blue 2-red: 488 FL1,2 640 FL3,44-blue: 488 FL1,2,3,4

FL1 FL2 FL3 FL4Standard: FITC PE PerCP-Cy5.5 APC2-blue 2-red: FITC PE APC-Cy7 APC4-blue: FITC PE PE-Cy5 PE-Cy7

Optical configurations

Detector Filter options Fluorophore

FL1 530/30 BP FITC, GFP, YFP, CFSE, AF-488

515/15 GFP

FL2 585/40 BP PE, PI

540/20 BP YFP

FL3 780/60 BP APC-eFluor-780, APC-Cy7

FL4 675/25 BP APC, PE-Cy5, AF-647

“2 blue 2 red”

Applications: BD™ CBA reagents

• BD CBA kits• Up to 7 analytes• Pre-defined panels

by area of biology• Th1/Th2• Th1/Th2/Th17• Inflammatory

cytokines• Chemokines• Anaphylatoxins

• BD CBA flex sets• Up to 30 analytes• User-defined panels

• Soluble proteins• Cytokines• Chemokines• Growth

Factors• Immunoglobulins• Enhanced

Sensitivity• <1.0pg/mL

Standard C6 configuration Selectable Lasers: 2-blue 2-red

Summary – The fluidics system

• Automatically starts up with the press of a single button.

• Self-cleans and alerts when fluids need attention.

• Is compatible with a variety of sample-tube configurations.

• Meters sample fluid uptake and cell counts

• Allows the user to independently adjust the speed of the sample uptake.

• Allows the user to adjust the core stream diameter for a range of cell sizes.

Summary – The optics system

• Is robust, making the instrument easy to transport.

• Allows the user to swap out interference filters easily.

• Requires no user alignment.

Summary – The electronics system

• Provides seven decades of dynamic range.

• Eliminates the need to adjust voltage settings.

• Provides software tools to help analyze a broad spectrum of data.

• Supports USB plug-and-play on a standard PC or laptop.

Summary – The software

• Can be used on any standard PC or laptop.

• Is intuitive to both novice and proficient users, even without training classes.

• Produces sophisticated graphics such as density plots and color histogram overlays.

• Uses the FCS 3.0 file format

• Allows users to drag and drop plot images into Microsoft Office applications

• and to copy and paste statistics to spreadsheet programs.

BD Accuri™ features that simplify multicolor analysis

• Digital system

• Four fluorescence detectors

• Flexibility in fluorochrome choice using:• Optional optical filters• Selectable Lasers option

• Predictable fluorescence spillover values

• Easy setup for data collection

• Validate system with beads

BD Accuri™ features that simplify multicolor analysis

www.bdbiosciences.com/support/resources/accuri

• Technical Bulletins

• BD Accuri C6 and BD Kits for Flow Cytometry

• BD Accuri C6 technical guidelines

• White papers

• BD Accuri proof sources

• User guides

• BD Accuri manuals

The BD Accuri™ C6 is Portable!

National Oceanic and Atmospheric Administration:Great Lakes Microcystis research project – Lake Erie

2nd Norwich Flow Day:Institute for Food Research

The Ecosystem Centre:Research vessel, Palmer Peninsula, Antarctica

Antarctica

Mobile Lab

Lake Erie