Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014
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Flow Cytometry 101: the “what, why and how” IMMU7040 - immunological Methodology February 18, 2014. Christine Zhang, PhD Faculty of Medicine University of Manitoba. Presentation Outline. Basic Concept of Flow Cytometry analysis and instrumentation

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Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014

Flow Cytometry 101: the “what, why and how”IMMU7040 - immunological Methodology February 18, 2014

Christine Zhang, PhD

Faculty of Medicine

University of Manitoba


Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014

Presentation Outline

  • Basic Concept of Flow Cytometry analysis and instrumentation

  • Applications of Flow Cytometry in Research Analysis

  • Fluorescence-Activated Cell Sorting (FACS)

  • Our Flow Cytometry Core Facility


Flow cytometry background

Flow Cytometry Background

What is it?

Emission light

Optical components

Detectors/amplifiers (PMT)

Excitation light

Lasers

Digital (computer/software)

Flow chamber and fluidics

 A system that integrates electronics, fluidics, optics, laser technology and computer analysis in a single platform.

 Allows for simultaneous multiparametric analysis of physical/chemical/biological characteristics of cells or particles at the single-cell level by detecting fluorescence intensityas they travel in suspension one by one past a sensing point

Mixed cells with fluorescence label


Flow cytometry background1

Flow Cytometry Background

What is it for?

  • Cell or particle size; cytoplasmic granularity

  • Cell surface antigens (immunophenotyping)

  • intracellular antigen (e.g. cytokine profiling)

  • Intracellular signalling (phospho-flow)

  • Cell viability/apoptosis

  • Cell cycle, DNA content/synthesis

  • Cell proliferation (BRDU and CFSE)

  • Intracellular Ca2+flux

  • Cell migration and adhesion

  • gene expression and transfer efficiency by reporter like GFP

  • mRNA expression in living cells

  • Protein-protein interaction by FRET

  • Activation sate and oxidative stress

  • Stem cell side population analysis

  • Cell sorting

I am a cell…

I am a virus…


The generation of fluorescent light

The Generation of Fluorescent Light

  • Electron of a fluorochrome absorbs light energy at certain wavelength (Ex)

    • Excited to a higher energy level S1 from ground level (S0).

    • Stair-step vibrational relaxation in picosec

    • Energy level returns to lower level and emit fluorescent light at certain wavelength (Em)

  • Different fluorochromes have distinct Ex/Em

Jablonski Diagram of Fluorescence in 1935


Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014

Variation in Ex/Em of Commencially Available Fluorophores

Excitation by lasers at

355nm laser (UV)

Emission Spectra of Alexa series

405nm laser (violet)

488nm laser (blue)

561nm laser (green)

633nm laser (red)

Each fluorochrome has its distinct excitation and emission spectra. Check if your instrument can excite and detect your fluorochromes first


Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014

Flow Cytometry: How does it work

Step 1: Panel Design—find the best marker-fluorophore combo for your instrument

Your Fluorophore Panel – fluorescence spectra viewer

  • Your Marker Panel

  • T cells: CD3+, CD90.2+

  • Effector T cell: CD4+, CD8+

  • Treg: CD25+

  • B cells: CD19+, B220+

  • Naïve B: CD27-CD138-

  • Plasma B: CD27+CD138+

  • Mono/Mac: CD14+CD16- or CD16+

  • NK: NK1.1+,CD56+

  • Neutrophils: CD11chiCD11bhiCD16hi

  • mDCs: Lin-MHCIIhi CD11chiCD123-

  • pDCs: Lin-MHCII- CD11c-CD123+

  • minimize Spillover: Choose fluorochromes excited by different lasers, avoid fluorochromes in adjacent channels

  • Be aware of the fluorochrome brightness index and marker expression: Bright fluorochrome – low expression marker; Dim fluorochrome – high expression marker


Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014

Flow Cytometry: How does it work

Step 1: Example of the Marker-Fluorochrome Panel Design

Goal: characterize IL-10 and TGFβ expression in Tregs in human PBMC

Key: Choose colors for low expressing-intracellular proteins for which staining is complicated first. Abundant easy-to-stain surface lineage markers last.

Marker Panel

CD4

CD25

Foxp3

TGFβ

IL-10

CD14

Fluorescence Parameters on FACSCanto

  • LaserFluorochrome:

  • blue (488 nm)FITC, GFP, Alexa488

  • PE

  • PerCP, PerCP-Cy5.5

  • PE-Cy7

  • red (633 nm)APC, Alexa647

  • APC-cy7

  • Violet (405 nm) Pacific blue, Dapi, V450

  • AmCyan, V500, BV510

My Panel Design

Fluorochrome Choice

BV510

PE-Cy7

APC

PE

Alexa488

eFluo450

Take-Home Message: Know your marks (e.g. expression, surface or intracellular) and fluorochromes (e.g. signal intensity, stability, etc.)


Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014

Flow Cytometry: How does it work (cont’d)

Step 2: Sample Preparation — fluorescence labeling proteins of your interest

GFP-vector transfected cellsFluorescence-tagged Ab labeled cells

Anti-protein BAb

Gene A

GFP

Gene B

RFP

Gene C

CFP

Anti-protein A Ab

Gene D

YFP

Protein A

Protein B

Remember to optimize your staining protocol: e.g. cell detachment, collagenase concentration, Antibody titration, staining buffer, suitable fix/perm solution, etc.


Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014

Flow Cytometry: How does it work (cont’d)

Step 2: Sample Preparation — fluorescence labeling proteins of your interest:

Cell Surface Staining

  • Collect cells and resuspend in PBS + 1% Fetal Calf Serum/BSA

  • Block FcγRs

  • Staining cells with primary antibodies-fluorochrome

  • Incubate on ice for 30 minutes to allow for antibody binding

  • Wash, optional fixation step, resuspend, analyze

    Intracellular staining/Phospho-Flow

  • Collect Cells as above

  • Block FcγRs

  • Fix and permeabilize cells

  • Rehydrate cells (preferred for phospho-flow analysis)

  • Staining with primary antibodies-fluorochrome

  • Incubate on ice for 30 minutes to allow for antibody binding

  • Wash, resuspend, analyze


Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014

Flow Cytometry: How does it work (cont’d)

Step 2: Sample Preparation — Know Your Cells

  • Cell Preparation

  • adherent cells need to trypsinize prior to staining  cell surface markers may be affected

  • large and sticky cells can be difficult to work with clumping, clogging the flow cell

  • Non-specific Staining:

  • FcR binding: can be blocked with antibody against FcR(Fc BLOCK)

  • Dead or dying cells: bind every fluorescently labeled antibody at a high level

  • Auto-fluorescence: some cells are intrinsically fluorescent without any staining at different level.

  • Autofluorescencetends to increase in all cells after fixation and prolonged storage

Macrophage: High autofluorescence

DC: Low autofluorescence


Flow cytometry how does it work cont d

Flow Cytometry: How does it work (cont’d)

Step 3: analysis on flow cytometer

FACSCalibur 2 laser system (4 fluorescence channels)

FACSCantoII 3 laser system (8 fluorescence channels)

LSRII 3 laser system (13 -15 fluorescence channels)


Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014

Inside the Flow Cytometer: Signal Generation

Sheath Flow

Sample Flow

Laser Beam

tube

sheath tank

Sample injection tube (SIT)

sheath filters/fluid lines

Collecting lens

Flow Cell

interrogation point

Waste tank

Flow chamber

1. Fluorescence-labeled Cells are carried to the flow chamber by liquid stream

2. Florochromes are excited by laser light beam

Sheath

Sample


Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014

Inside the Flow Cytometer: Signal Generation

3. Emitted fluorescent light pass through a set of optical filters

4. Light signals are amplified and detected by detectors (PMT).

5. Light signals are converted to digital signals and sent to computer for analysis

PMT

LP filter

PerCP

BP filter

APC-Cy7

PE

SSC

FITC

PE-Cy7

APC

PE-TR


Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014

Understanding the Optical Components: Longpass Filters and Bandpass Filters

3) PMT

Longpass Filter

APC-Cy7

% of transmission

2) BP filter

1) LP filter

Wavelength (nm)

BandpassFIlter

APC

% of transmission

3) PMT

Wavelength (nm)

How to read a longpass filter and bandpass filter detection range

e.g.: 502LP  wavelength longer than 502nm will be allowed to pass through

e.g. 530/30 = 530nm +/- (30/2)nm = 530nm +/- 15nm = 515nm – 545nm


Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014

Flow Cytometry: Parameters

  • Forward Scatter: measures cell or particle size

  • Side Scatter: measures internal complexity or granularity of the cell

Example of Fluorescence Parameters on LSR-II

  • 3 Lasers, 16 parameters (Forward Scatter, Side Scatter, 14 fluorescent detectors)

  • LaserMirror/FilterFluorochrome

  • blue (488 nm)505LP, 530/30FITC, GFP, Alexa488

  • 550LP, 575/25PE, dsRed, Alexa546, Cy3

  • 600LP, 610/20PE-Texas Red, PI (live)

  • 635LP, 670/14PE-Cy5.5, 7-AAD

  • 735LP, 780/60PE-Cy7

  • red (633 nm) 660/20APC, Alexa647, Cy5

  • 710LP, 730/45Alexa700

  • 755LP, 780/60APC-cy7, APC-Alexa750

  • Violet (405 nm) 450/40Pacific blue, Dapi, V450

  • 475LP, 525/50AmCyan, CFP, V500

  • 545LP, 560/20Qdot 565

  • 575LP, 585/15Qdot 585

  • 595LP, 605/12Qdot 605, BrilliantViolet605

  • 630LP, 655/8Qdot 655


Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014

Flow Cytometry can detect cells or particles with a wide range of size

Size beads only (0.1 – 1.1μm)

Viral particles

Cell-derived microparticles

Primary mouse splenocytes

Granularity (side scatter)

size (forward scatter)


Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014

1.

2.

3.

Laser

Voltage

time

Voltage

Laser

time

Laser

time

Flow Cytometry: Signal Conversion in PMT

Photon  Current  Voltage  Digital Signal

  • Fluorescentemissionsaredetectedas a voltage pulse fromphotomultipliertube (PMT) detectors

  • The area, voltageandheightofthevoltage pulse ismeasured

Voltage


Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014

Flow Cytometry: Data Analysis

  • Flow cytometry data can be plotted in several different ways:

  • the axes of the graphs represent fluorescence intensity data, usually plotted on a log scale

  • for histograms, the y axis is cell number

Histograms


Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014

Flow Cytometry Analysis – from Simple to Complex

1 color flow (e.g transfection efficiency or 1 color staining)

Monoclonal stable cell line

More Granular

Dead Cells and debris

Bigger Cells

GFP expression

Whole blood

Side Scatter (SSC)

GFP+: 87.5%

smaller cells

Live Cells

Receptor A

Receptor B

cell count

Bigger

Forward Light Scatter (FSC)

SSC

FSC

GFP (MFI)

Brighter


Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014

Flow Cytometry Analysis – from Simple to Complex

9 color Flow (e.g. immunophenotyping and clinical diagnosis)

Double Positive Population

Single PE Positive Population

Negative Population

CD8-PE

CD4-FITC

Single FITC Positive Population

Wood B. 2006. Arch Pathol Lab Med. 130:680–690


Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014

Be Aware of signal spill-over for large panel analysis

  • Fluorescence signal spill over = signal from one fluorochrome (e.g. FITC) being picked up by detector for another fluorochrome (e.g. PE).

  • For compensation, use single stained controls for every fluorochromeyou use along with unstained control

FITC only

Before compensation

unstained

Compensation to remove signal spillover

after compensation

PE - %FITC  take out x% of the signal in PE that is due to spill-over from FITC


Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014

Signal Spillover and Color Compensation

  • Tips on compensation:

  • Compensation is specific for fluorochromes, NOT for cell types  compensation values are valid for all cell types

  • Try Compensation beads if your cell samples are precious or if your marker expression is low.

  • Choose good markers that gives sharp clear positive peak away from negative peak

    • lymphocytes: CD4, CD8, CD90, CD19. DC/neutrophils: Gr-1, CD11b)

  • Expression should be equal or higher than the experimental samples

  • Minimize spillover by spreading your colors of choice over different lasers and avoid adjacent channels

  • Manual compensation to double check

uncompensated

compensated

over compensated

under compensated

Median values both = ~3.2


Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014

Western Blotting vsFluorescence Microscopy vs Flow Cytometry


Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014

Presentation Outline

  • Basic Concept of Flow Cytometry analysis and instrumentation

  • Applications of Flow Cytometry in Research Analysis

  • Fluorescence-Activated Cell Sorting (FACS)

  • Our Flow Cytometry Core Facility


Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014

Flow Cytometry Application

Immunophenotyping and Cytokine Profiling – human PBMC

Workflow

Maecker H. Nature Reviews Immunology 12, 191-200


Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014

Immunophenotyping and Cytokine Profiling – human PBMC

  • Marker Panel

  • T cells: CD3+, CD90.2+

  • Effector T cell: CD4+, CD8+

  • Treg: CD25+

  • B cells: CD19+, B220+

  • Naïve B: CD27-CD138-

  • Plasma B: CD27+CD138+

  • NK: NK1.1+,CD56+

  • Mono/Mac: Lin-CD14+

  • Classical: CD16-

  • Non-classical: CD16+

  • mDCs: Lin-HLA-DRhi CD11chiCD123-

  • pDCs: Lin-HLA-DR- CD11c-CD123+

Cytokine profiling on the desired population:

e.g. IFNα, IL10, CXCR3, CCR5, TFNα

Maecker H. Nature Reviews Immunology 12, 191-200


Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014

Immunophenotyping and Cytokine Profiling – human PBMC

10-color analysis on moncytes subsets (viability marker, lineage markers and cytokines all in 1 tube)

Exclude doublets

Exclude the dead

PMBCs

Exclude T, B, NK

CXCR3

CCR2

CD16

CD14

TNFα

IL10

Cytokine profiling

3 Monocyte subsets

Exclude HLA-DR neg


Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014

Immunophenotyping – B Cell Leukemia Diagnosis by Flow Cytometry

  • B-cell neoplasia(e.g. CLL) can be diagnosed by flow cytometry, WBC count and clinical history.

  • more than 5x109 monoclonal B cells in the blood  CLL

  • Elevated B cell number alone is not enough to diagnose various subclasses of B-Cell Neoplasia

>40% CD19+ cells in PBMC

CD19

CD23

CD11c

CD5

CD5

CD5

CD5

CD19

CD23

CD19

CD19

CD5

CD20

Chronic Lymphocyte Leukemia (CLL)

Mantle Lymphocyte Leukemia (MCL)

Hairy Cell Leukemia (HCL)

CD19+CD5+CD23+

CD19+CD5-CD20+CD11c+

CD19+CD5+CD23-

MaryaliceStetler-Stevenson, Flow Cytometry Unit, NIH


Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014

Flow Cytometry Application

Receptor Signaling Cascade by Phospho-Flow

Cell Surface Staining

  • Collect cells

  • Block FcγRs

  • Staining cells with primary antibodies-fluorochrome

  • Wash, resuspend, analyze

    Intracellular staining/Phospho-Flow

  • Collect Cells

  • Block FcγRs

  • Fix and permealize cells

  • Rehydrate cells (preferred for phospho-flow analysis)

  • Staining with primary antibodies-fluorochrome

  • Wash, resuspend, analyze


Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014

Receptor Signaling Cascade by Phospho-Flow – human PBMC

Key Ingredient: good phospho-specific antibodies directly conjugated to fluorophore

Note: phosflow is limited only to high abundance protein that are highly phosphorylated on very unique sites that can be detected with specific antibodies (a small subset of signaling

molecules)

Krutzik P. 2011. Flow Cytometry Protocol; Chapter 9, Fig. 2


Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014

Flow Cytometry Application

DNA Content and Cell Cycle Analysis

Cell Cycle Phases

DNA histogram

G0/G1 phase (2N)

S phase (2 - 4N)

Debris

G2/M phase (4N)

Common dyes that bind stoichimetrically to DNA: Propidium Iodide, Hoechst 33342, DAPI, 7AAD ,DRAQ5, etc

Tabll A. 2011. Liver Biopsy; Chapter 7, Fig. 2


Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014

Flow cytometry-based Cell Cycle Analysis in human breast cancer

Normal cell (diploid)

G0/G1 = 60%

S = 13%

G2/M = 27%

Diploid

tetraploid

Tumor cell (aneuploidy)

G0/G1 = 79.5%

S = 12.7%

hyperdiploid

G0/G1

G2/M = 7.8%

Tumor

Diploid

hypertetraploid

Count

Count

G2/M

S

DNA CONTENT

DNA CONTENT

Ross J. 2003. Am J ClinPathol. 120: S72-S84


Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014

Flow Cytometry Application

Cell Viability and Apoptosis

31% later stage

Membrane blabbing

Apoptotic bodies

Nucleus collapse

Cell dehydration

Chromatin condensation

Loss of PS asymmetry

Apoptosis

Propidiumiodide

35% early stage

33% Viable

Annexin V (phosphatidyl serine)


Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014

Cell Viability and Apoptosis

  • Early and late apoptosis (programmed cell death) can be measured based on several different type of cellular alterations

  • Each type of alteration can be detected by flow

    1) Activation of caspases:detected by staining with antibodies detecting cleaved caspases

    or caspase substrates OR staining with fluorescently-labeled caspase inhibitors (Casp-Glow)

    2) Mitochondrial dysfunction:detect changes in membrane potential with Rhodamine 123,

    TMRE, MitoTracker dyes or cytochrome C release using specific antibodies

    3) Alterations in membrane symmetry:phosphatidyl serine translocates from cytoplasmic

    to extracellular side of membrane > detected by annexin V binding (note: membrane inversion also occurs during granule release in neutrophils, mast cells, etc)

    4) Loss of membrane integrity: apoptotic cells become permeant to DNA-binding

    dyes such as DAPI or PI

    5) DNA fragmentation: TUNEL assay > TdT enzymatic incorporation of fluorescent

    nucleotide analogues


Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014

Flow Cytometry Application

Cell Proliferation and DNA Replication by BrdU or CFSE

BrdU/EdU: incorporated into the newly synthesized DNA of replicating cells (during the S phase)

CSFE: measure cell division as CSFE fluorescence intensity is halved within daughter cells after each cell division

BrdU

count

EdU

low-proliferating BM cells

Highly-Proliferating atrial cells

Propidium Iodide

Non-proliferating cells

Proliferating cells


Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014

Presentation Outline

  • Basic Concept of Flow Cytometry analysis and instrumentation

  • Applications of Flow Cytometry in Research Analysis

  • Fluorescence-Activated Cell Sorting (FACS)

  • Our Flow Cytometry Core Facility

FACSAriaIII 3 laser system (15-16 fluorescence channels)


Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014

Fluorescence-Activated Cell Sorting (FACS)

  • FACS:a specialized type of flow cytometry to sort a heterogeneous mixture of cell suspension

  • Features

  • Sort up to 4 populations of interests

  • 15 fluorescence color simultaneous on the same cell

  • Sort different types cells

    • Primary BM, PBMC, mouse splenocytes

    • Any types of cell lines

    • Large fragile cells like activated neutrophiles, lung DCs

    • Sticky and hard to sort cells (e.g. solid tumor cells, neuron cells)

  • Multi-purpose sorting

    • 7ml round bottom tube, 15ml conical tubes.

    • Tissue culture plates, 96 well PCR plates

    • Microscope slides including multiwellchamber slides

    • Single cell sorting

    • Different modes to maximize sort purity (99% for qPCR) or recovery (for assays requiring large number of cells

  • sterile sorting, sample agitation, temperature control

Mixture of cells to be sorted

laser

PMT

nozzle

+

New drop

empty drop

_

_

+

_

+

_

_

+

_

+

+

_

+

_

+

_

_

+

+

+

_

+

v


Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014

FACS cell soring pros and cons

Mixture of cells to be sorted

  • FACS sorting

  • Pros:

  • Good for sorting very rare population or any populations of interests.

  • Accommodate large panel of markers (up to 15 colors)

  • Sort up 4 populations simultaneously

  • Cell sample quality check by flow cytometry before sorting.

  • High purity, low death rate after sorting

  • Cons:

  • Require specialized instrument and dedicated personnel

  • May take longer to sort a large number of cells

laser

PMT

nozzle

+

New drop

empty drop

_

_

+

_

+

_

_

+

_

+

+

_

+

_

+

_

_

+

+

+

_

+

v


Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014

Sorting Example 1: Enrichment of Ramos Cells transiently transfected with 4 different constructs

Before Sorting

SHIP

PD

EGFP

Y944F

After Sorting

Count

GFP


Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014

Sorting Example 2: sort mature and immature neutrophils from mouse BM

ma neu

Before Sorting

After Sorting

imneu

SSC

FSC

CD11b

Gr-1


Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014

Our Instruments – Flow Cytometry Analyzers

BD FACSCanto-II Digital Flow CytometryAnalyzer

Location: Room 466, Apotex Center, University of Manitoba BannatyneCampus

3 Lasers: 1) 488 nm blue laser; 2) 633 nm red laser; 3) 405 nm violet laser

10 parameters: FSC, SSC, 4 fluorescent detectors off 488 nm, 2 fluorescent detectors off 633 nm, 2 fluorescent parameters off 405 nm.

BD LSR-II Digital Flow Cytometry Analyzer

Location: Room 536, Basic Medical Science Building, University of Manitoba BannatyneCampus

3 Lasers: 1) 488 nm blue laser; 2) 633 nm red laser; 3) 405 nm violet laser

16 parameters: FSC, SSC, 5 fluorescent detectors off 488 nm, 3 fluorescent detectors off 633 nm, 6 fluorescent parameters off 405 nm


Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014

Our Instruments – FACS sorter

BD FACSAriaIIIDigital Cell Sorter

Location: Room 462, Apotex Center, University of Manitoba BannatyneCampus.

3 Lasers: 1) 488 nm blue laser; 2) 633 nm red laser; 3) 405 nm violet laser

17 parameters: Forward Scatter, Side Scatter, 6 fluorescent detectors off 488 nm, 3 fluorescent detectors off 633 nm, 6 fluorescent parameters off 405 nm

Features: sample agitation; temperature control; two-way or four-way sorting into tubes, multiwell plates or microscope slides; Aerosol Management Option (AMO); housed in a ClassII Type A2 biosafety cabinet; accommodate most cell types.


Flow cytometry 101 the what why and how immu7040 immunological methodology february 18 2014

My Contact

Christine Zhang, Ph.D.

Flow Cytometry Core Facility Manager

Faculty of Medicine, University of Manitoba

413 Apotex Center, 750 McDermotAve.

Tel: (204) 294-0691

Email: [email protected]

http://umanitobaflow.ca/

Useful Resources

http://www.cyto.purdue.edu/flowcyt/educate.htm

http://www.lifetechnologies.com/ca/en/home/life-science/cell-analysis/flow-cytometry/flow-cytometry-technical-resources.html

http://www.bdbiosciences.com/research/multicolor/spectrum_viewer/


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