Principle of Flow Cytometry · Accuri Innovations in Flow Cytometry • Innovations in all the...

®

A powerful system with a simple design.

產品應用專員陳思穎

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Principle of Flow Cytometry

Microscopy

• qualitative• Slow• Statistics?

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Why we use flow cytometer?

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Hydrodynamic Focusing

Bernas T, Grégori G, Asem EK, Robinson JP.Mol Cell Proteomics. 2006 Jan;5(1):2-13. Epub 2005 Oct 28. Review.

Printed with FinePrint trial version - purchase at www.fineprint.com

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Bernas T, Grégori G, Asem EK, Robinson JP.Mol Cell Proteomics. 2006 Jan;5(1):2-13. Epub 2005 Oct 28. Review.

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Forward Scatter (FSC) & Side Scatter (SSC)

Light source

Side scatter detector

Forward scatter detector

Lymphocyte Gate

CD3-APC

CD

4-PE

Gate:CD3+ CD4+

• Over 7 Decade dynamic range:Eliminating the need to set gain or voltage reduces the consumption of valuable sample and sheath during set-up

• Absolute counts:It is simple to obtain absolute counts, or sample concentration per microliter without the need to add beads to samples

• Automated CSampler®

Standard 96-well (flat, round, and v bottom) Plates, deep-Well 96-well Plates, 48-well Plates, 24-tube rack (for 12x75 mm tube) .<90 minutes for 96-well plate, utilizing 30 second acquisition time per well.

• Use DI water as sheath fluid• CFlow software is easy to learn: zoom in function• Compact size, simple maintenance

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Leading Advantages of Accuri C6

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The Accuri C6 is Robust!

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

2nd Norwich Flow Day:Back of Kate’s car, Institute for Food Research

The Ecosystem Centre:Palmer Peninsula, Antarctica


Accuri Innovations in Flow Cytometry

• Innovations in all the major components of a flow cytometer

o Fluidics: allows direct-volume measurement

o Optics: locked-down alignment

o Signal detection: broad dynamic range obviates voltage adjustments

o Software: developed by “high tech anthropologists” trained to facilitate human-computer interactions

• New and modern approaches exploited wherever possible

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Accuri Innovation - Fluidics

• Laminar flow fluidics• Non-pressurized, peristaltic pump-

driven system• Patented pulse dampeners• User controls both flow rate and core

diameter• Volume measurement for absolute

counts• Minimum sample volume 25 µL• Up to 10,000 events/secondSheathSheath

PurgePurge

WasteWaste

Flow CellFlow CellLaserLaser

Sam

ple

Sam

ple

Det

ecto

rD

etec

tor

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Unique Fluidics System Simplifies Sample Handling

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• Microprocessor-controlled peristaltic pumps enable direct volume measurement

• Many types of sample tubes may be used• No need to transfer samples • Save time and materials• Add reagents or cells during a run• With CSampler: culture, stain and run, all in one

plate

Cell Count Verification for the Accuri C6

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y = 1.116xr² = 0.9774

n = 75

1,000

10,000

100,000

1,000,000

10,000,000

1,000 10,000 100,000 1,000,000 10,000,000

Cou

ntin

g B

ead:

cel

l cou

nt p

er m

L

Direct Volume: cell count per mL sampleCell Counting Method

1 2 3

Coe

ffici

ent o

f Var

iatio

n

0

10

20

30

40

direct-volume(C6 cytometer)

counting beads(C6 cytometer)

Hemacytometer

Accuri C6 Counting beadsp= 0.01

Hemacytometerp = 0.002

The average coefficient of variation for replicate cell counts using three different counting methods on the same samples.

A paired student’s T test was used to determine p values (95% confidence, N = 23).









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FSC

SSC

Accuri Innovation –Pre-optimized and locked-down optics

Compact optical system design reduces cost and

eliminates alignment issues

510/15540/20565/20610/20780/60

User changeable optical filters

488 nm solid state laser

640 nm diode laser

PMTs for fluorescenceDetection

Diodes for scatter detection

FL1 530 / 30nm (FITC/GFP)FL2 585 / 40nm (PE/PI)FL3 >670nm LP (PE-Cy5, PE-Cy5.5, PerCP-Cy5.5, PE-Cy5, PE-Cy7)FL4 675 / 25nm (APC)

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C6 optics layout:No dichroics, co-linear lasers








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FL4 = 675/25

Instrument-to-instrument variation is minimal

FL3 = 670LP

6-Peak APC (FL4)

8-peak data from multiple C6 instruments manufactured over a six-month period.

Accuri C6 Detectors are Characterized and Balanced at Manufacture

FL1 = 530/30 FL2 = 585/40

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

Beads: 1, 2, 12, 29 micron

“A GFP signal that was off scale on our other flow cytometer gave us a distribution that was contained within the 24-bit scale on the C6.”

Ian Dimmick, Flow Cytometry Core Facility Manager, Institute of Human GeneticsNewcastle University, UK

Zoom Area

Zoom Area

Accuri Innovation –Electronics with Broad Dynamic Range

More than 6 decades of signal on a single scaleAll your data, available at any time

18-Bit System

24-bit Accuri C6

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6+ log view

24 bit18 bit

5 log view4 log view

8 bit

8-Peak Validation Beads

More than 6 logs of dynamic range,24-bit digital signal processing

Zoom

4 log view

24 bit

A wide range of particle sizes are on scale

Beads: 1, 2, 12, 29 micron

Zoom Area

Zoom Area


How does one deal with all that resolution?

6+ log view

Zoom

Human peripheral blood

Zoom

Un-Zoom

HPB Lymph Gate

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Advantages of Pre-optimzed Detector Settings

• 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 spill-over

• Focus on the science of measuring fluorescence, not the art of setting voltages








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Lymphocytes

Zoom Area

Zoom OutZoom Again

Pre-Optimized Light Scatter DetectorsAdjust Your View with Zoom

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Pre-optimized Light Scatter DetectorsAdjust Your View With Zoom

Accuri Innovation – Intuitive Software

Sample Grid

CytometerStatus

Fluidics Controls

Run Criteria

Threshold and Compensation

Real Time Updates

Analysis and Gating Tools

Regions

Quads

Markers

Histograms

Dot plots

Density plots

Plot Statistics

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• Selectable Laser Module

2-blue 2-red: FL1 and FL2 read blue laser-excited emissions;FL3 and FL4 read red laser-excited emissions.

4-blue: All 4 detectors read blue laser-excited emissions.

• CSampler®

• FiltersAttenuation filters and optical filters

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Other Modules and Accessories

510/15540/20565/20610/20780/60

90 ％99 ％

CSampler®

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Optional Filters and Selectable Lasers Provide Flexibility

Separation of GFP and YFP signals

FL1: 530/30 FL1: 510/15

Standard Filters GFP /YFP Combo

FL2:

585

/40

FL2:

540

/15

GFPYFP

YFP

GFP

Selectable Lasers:Reassign laser and detector associationsStandard: 488 FL1,2,3 640 FL42-blue 2-red: 488 FL1,2 640 FL3,44-blue: 488 FL1,2,3,4

CD3+CD4+

CD3-APC-Cy7

CD

4-A

PC

CD45RA-FITC

CD

45R

O-P

E

CD45-RA - FITC


• Add-on automation for the C6 Flow Cytometer

• Processes 96-well and 48-well plates

• 24-position rack for 12 x 75 mm tubes

• Priority interrupt for urgent samples

• Easy-to-use software

CSampler®

Automated Flow Cytometry. Now It’s Easy.

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Screening? Consider the CSampler®

• Add-on automation for the C6 Flow Cytometer

• Processes 96-well and 48-well plates• 24-position rack for 12 x 75 mm tubes• Priority interrupt for urgent samples• Easy-to-use software

62,421

4,845

Proportion: 4,84562,421

= .077

Define fluorescence spill-over

FITCemission intensity

wavelength

585 BP530 BP

Calculatemedians

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Pre- and post-compensation


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Spillover in 1-D: CD45-PE-Cy7+

Spillover not corrected

Spillover corrected

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Predictable Spillover Allows Average Values to be Defined

Fluorochrome Spillover

Slope ofgraphed data

Average Compensation

FITC into FL2 0.0770 7.00%

PE into FL1 0.0370 3.50%

PE‐Cy7 into FL4 0.0013 0.00%

PE‐Cy5 into FL4 0.4613 47.00%

APC into FL3 0.0075 1.00%

Suggested Compensation Values for the Accuri C6

FITC PE PerCP PerCPCy5.5 PE-Cy7 APCFL1 (530BP) --- 3.5 0.00 0.0 1.00 ---

FL2 (585 BP) 7.0 --- 0.00 0.00 3.50 0.0

FL3 (670 LP) 1.0 14.5 --- --- --- 1.2

FL4 (675 BP) 0.0 0.0 3.00 12.00 0.00 ---

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Applications

PI - DNAB

RD

U F

ITC

• Standard 4 color immunofluorescence analysis• Cellular proliferation and tracking with cell tracker dyes

• Cell cycle analysis

• Multiplex bead analysis: Cytokines

• Dynamic cell processes: Ca2+ flux

• Protein expression and screening: siRNA, transfection, infection

R2Bea

d in

tens

ity

Analyte intensityCFSE

FSC

-A

Exploring the Research Power of the C6

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No virusLuciferasePyk2

GFP Expression


Lymphocyte Gate

Human PBMC : T cell Phenotyping

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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 cells only 0.4%

DC + T cells1 : 4 52.4%

In Vitro T cell proliferation: T cells + dendritic cells (DC)

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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

DNA Detection and Cell Cycle Analysis

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Data File Format:• FCS 3.0 compliant

Compatible with:• FCS Express: CFlow File

Importer• MultiCycle• FlowJo 7.6• WinlistTM and Modfit LTTM

• VenturiOne®

Calcium Flux Measurement with Fluo-4

Sample tubes on the C6 do not require pressurization. Agonists can be added during sample acquisition, ensuring that one is able to visualize the entire kinetic activity.

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Multiplex Bead AnalysisAssay Designs HSP/Chaperone 8-Plex MultiBead™ Kit

Methods: Data from Assay Designs™ multiplex bead immunoassay kit for the measurement of HSP client proteins (Akt and Akt pSer473) and HSPs (Hsp27 pSer15, Hsp27 pSer82, Hsp40, Hsp60, Hsp70 and Hsp90 alpha) in cell lysates. Plots show results from a non-heated control and a heat treated hela-2 cell lysate. Standard curves and quantitative analyte levels can be obtained using the MultiBead software provided by Assay Designs.

Inhe

rent

Bea

d Fl

uore

scen

ce

Analyte intensity Analyte intensity

Inhe

rent

Bea

d Fl

uore

scen

ce(6) (6)

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Luc

α-Pyk2

α-actin

Pyk2NV

A.

B.

Monitor Suppression of Pyk2 Expression by microRNA-containing Viruses

Methods: Non-adherent,human PBMCs werestimulated for 3 days with anti-CD3 and anti-CD28 antibodies.The resultant cells were 99%pure for CD4 and CD8expression (APBT). APBTswere uninfected (No virus/NV)or infected with 2 MOI of alentivirus that contained GFPand a microRNA specific forLucifierase (luc) or Pyk2(Pyk2).

Results: A) The expression ofGFP was assessed by flowcytometry. B) The effects ofvirus infection on Pyk2 andactin expression was assessedby immunoblotting.

No virusLuciferase

Pyk2

GFP Expression

Fluorescent Protein Detection with the Accuri C6

Transfection Screening

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gp64-PE Expression in SF9 Insect Cells Infected with Baculovirus.

P2

P3

Virus Generation

ViralDilution

No virus 1:10 1:100 1:1000

gp64

-PE

Gp6

4-PE

gp64

-PE

FSC-H


White Cell Differential: Single Platform Cell Counts

PopulationCD45 vs. SSC

GateCell

NumberVolume

Pulled (L)Sample

Volume (L) Cells x103/mLNormalRange

Lymphocytes P5 60,827 308.1 2,100 4.15 0.8 - 5.0

Monocytes P6 1,154 308.1 2,100 0.08 0.1 - 1.0

Granulocytes P7 26,618 308.1 2,100 1.81 1.4 - 7.5

Eosinophils P3 2,478 308.1 2,100 0.17 0.0 - 0.4

Calculation of cell number per mL of original blood sample for four identified populations.

FSC vs. SSC CD45 vs. SSC CD45 vs. SSC

Platelets

Lymphocytes

Monocytes

GranulocytesEosinophils

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DNA Staining with Propidium Iodide

Methods: Jurkat cells were treated with either DMSO (Control; <2%) or HU-331, an apoptosis inducing cannabinoid (5 g/mL), Cayman Chemical Company, for 6 hours. Cells were then fixed and stained with Accuri Cell Cycle Phase Determination Kit (KR-300).

Control (DMSO) HU-331

G1

S G2/M

Sub - G1

Simultaneous Analysis of GFP-Transgene Expression and Plant Cell Ploidy

2C

4C8C

16C

Analysis of wild type A. thaliana root nuclei, showing initial PI vs. SSC gate and polyploid populations (2C through 16C).

Data courtesy of Dr. David Galbraith, University of Arizona, Department of Plant Sciences, Tucson, AZ, USA

Analysis of wild type (WT) and GFP transgenic A. thaliana root nuclei. GFP-transgene expression is concentrated in nuclei with greater than 4C DNA content.

GFP

Log PILog PI

GFP

WT GFP Transgenic

2C 4C 8C 16C

2C 4C 8C 16C

Image expanded using Zoom tool

Use of 7-AAD for Viability Determination

7-AAD

Viable cells within higher FSC region are

impermeable to 7-AAD

Methods: Splenocytes from 3 to 6-month old C57BL/6 mice were irradiated with UV light. Cell viability was determined by adding 1 µL 7-AAD from a stock solution of 1 mg/mL (BD PharmingenTM) per 100 µL volume of cells.

Dead cells within lower FSC region are

permeable to 7-AAD


CD8a PE‐ALight Scatter Gate

Gate = P1

CD8a PE-A

7-A

AD

Viable T Cells: Lower Right Quadrant

Count Volume ( μL) Cells/µL PhenotypeGated on (P1 in all)

This Plot 58,588 6.4 9154 Total Cells in P1Q8-UL 4,354 6.4Q8-UR 243 6.4Q8-LL 48,088 6.4

Q8-LR 5,903 6.4 922 Viable CD8+ Cells

Gate = P1

CD4 FITC-A

7-A

AD Count Volume ( μL) Cells/µL Phenotype

Gated on (P1 in all)This Plot 58,588 6.4 9154 Total Cells in P1Q9-UL 4,495 6.4Q9-UR 571 6.4Q9-LL 45,647 6.4

Q9-LR 7,875 6.4 1230 Viable CD4+ Cells

Absolute counts of viable CD8 and CD4 T cells:Mouse splenocytes

TUNEL Alexa-488

CD

68 A

lexa

-647

Phagocytosis by Macrophages

Macrophages + Thymocytes

Courtesy of James Shayman, M.D. Department of Nephrology, University of Michigan, Ann Arbor, MI, USA

CD

68 A

lexa

-647

TUNEL Alexa-488

Macrophages Thymocytes (damaged)

TUNEL Alexa-488

CD

68 A

lexa

-647

C6 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 mixtureE. coli B

+ GFP plasmidE. coli B wild type

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

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Compensation: why bother?human peripheral blood lymphocytes


Advantages of the Accuri C6 innovations

Fluidics • Non-pressurized• Any sample tube• Continual calcium flux measurements• Absolute cell counting

Software • Intuitive and easy to use• Zoom tool• Time as a parameter• FCS 3.0 compliant

Optics• Pre-optimized detectors• Fixed optical alignment• Reduced training and instrument setup time• Reduced maintenance • Simpler QC/reduced variability

Electronics• No voltage or gain settings• ALL data collected from every channel all the time• Less sample and time used for instrument setup • Predictable fluorescence spill-over • Simpler QC/reduced variability

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Thank you! Any questions?


Principle of Flow Cytometry · Accuri Innovations in Flow Cytometry • Innovations in all the...

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