Yan Deng (X4-5225), ydeng@bu.edu

Gerald Denis (X4-1371), gdenis@bu.edu

Mike Xie (X4-5225), mike.xie@bmc.org

for users of theFlow Cytometry Core Facility

at BUMC

21 September 2010

Introduction to the Principlesof Flow Cytometry

John Meyers (X8-7543), johnm@bu.edu

Flow Cytometry Core Facility: Personnel

Director of Cell Sorting Services: Yanhui Deng

Operations Manager: Mike Xie

Facility Consultant: John Meyers

Co-Director/Outreach: Gerald Denis

Director: David Sherr

http://www.bu.edu/cores/flow-cytometry

Where are the instruments located?

LSR II,MoFlo:670-5

FACScan:R901

FACScan:X620

FACScan,FACSCalibur:L50

8

http://www.bu.edu/cores/flow-cytometry

U R here

Flow cytometry was not easy in the old days

gendered division of labor

ungloved handsmissing PPE

live anthraxbacilli

But today… it’s as easy as PCR.

fluidics

optics

ele

ctro

nic

s3 elements of any

flow cytometry system

fluidics

FluidicsPurpose of the fluidics system:

1. Transport particles in a fluid stream to the laser beam to be interrogated

2. Position the sample core in the center of the laser beam

sheath fluid

samplefluid

‘hydrodynamic focusing’

single file particles

● low flow rate

● narrow sample core

● high resolution

● high flow rate

● wide sample core

● low resolution

Always filter your samples to remove aggregates.

Fluidics

When conditions are right (i.e. when turbulence is minimal):

sample fluid flows in a central core

does not mix with the sheath fluid

This is termed ‘laminar flow’

optics

“SSC”

“FSC”

ele

ctro

nic

s

Forward and side scatter of leukemic cellsMalignant, large B cellsNormal B cells

FSC

SSC

The intensity of forward scatter light is proportional to size and cross-sectional area of the cells.The intensity of side scatter light is proportional tosize, shape and internal structure/irregularity of the cells.

Photomultiplier (PMT) detectors convert photons (selected by mirrors and filters) to electrical

pulsesPeak Height (volts)

Peak Width (time)

Peak Area

The higher the PMT voltage (user controllable), the greater the output magnitude for a given photon. At higher PMT voltages, the level of noise will also increase.

Adjusting the voltageof the PMT helps tooptimize captureof desired populations

Photomultiplier Tubes (PMTs)

FSC: forward scatter(size; cross-sectional area)

SSC: side scatter (granularity, internalor surface structurethat scatters light)

A dotplot represents two properties of a single cell

0 50K 100K 150K 200K 250KFSC-A

0

50K

100K

150K

200K

250K

SS

C-A

FSC

SSC

A histogram represents the distribution of a single parameter across many cells

0 102 103 104 105

APC-A: pH2AX

0

20

40

60

80

100

% o

f Ma

x

Control Condition Experimental10,000 cells each!

1 cell

1 cell

1 cell1 cell

Electronic processing of emission signals

Amplifiers are of two types: linear or logarithmic

Linear amplification is typically used with scatter.

Logarithmic amplification is typically used with fluorescence.

DNA content (Linear detection)

DNA content (Log detection)

Gating

0 50K 100K 150K 200K 250KFSC-A

0

50K

100K

150K

200K

250K

SS

C-A

0 102 103 104 105

APC-A: pH2AX

0

50

100

150

# C

ells

Gating allows one to select populations based on computer or human-derived criteria and further gate or display the included

cells

Backgating – don’t lose your bearings!

Backgating allows one to determine if a gating strategy is all-inclusive of a desired cell type.

In the above example, some cells are missed! What are they?Many investigators overlook the importance of verification by backgating!

FLUORESCENCE

Excitation wavelength and emission wavelength areunique properties of each specific molecular structure

(FITC)

blue laser

Stokes Fluorescence

Excitation Emission

Stokes shift

Fluorescein (FITC)

Hoechst 33258 Texas Red

Propidium iodide (PI)

Laser light must overlap with excitation wavelength

yes

no

488

488

488

488

ex

ex ex

ex

em

em em

em

But different lasers are available to excite other molecules (LSR II)

488 nm (Blue) : FITC, GFP, PE, PerCP, PE-Cy5, PI, PerCP-Cy5.5, PE-Cy7

Widely-used molecules are excited by the 488 nm laser (FACScan)

355 nm (UV) : Indo-1, DAPI, Alexa Fluor 350, Hoechst 33258405 nm (Violet) : Alexa Fluor 430, Alexa Fluor 405, Pacific Blue 561 nm (Yellow/Green): Texas Red, Cherry Red, Tomato Red633 nm (Red): APC, APC-Cy7, Alexa Fluor 647, Alexa 680

bandpass filters

longpass dichroic mirrors

Octagon Detector Arrays

emitted fluorescent light

EMISSION

weakly expressedepitope

strongly expressedepitope

Fluorescence detection

Note logarithmic scale

autofluorescence

weakly expressedepitope

Isotype controls

Isotype control antibodies should be used at the same concentration to stain cells at the same cell density as the experimental, but they give fluorescent signals that define a negative result.

How do you knowit’s real?

isotype control test

Resolution sensitivity

Resolution sensitivity, the abilityto resolve a faint signal frombackground) depends on thedifference D between the positiveand background peaks and the spread of the backgroundpeak W

Reagent Stain Index

Phycoerythrin (PE) 356.3Alexa Fluor 647 313.1

APC 279.2PE-Cy7 278.5PE-Cy5 222.1

PerCP-Cy5.5 92.7PE-Alexa Fluor 610 80.4Alexa Fluor 488 75.4FITC 68.9PerCP 64.4APC-Cy7 42.2Alexa Fluor 700 39.9Pacific Blue 22.5AmCyan 20.2

Choose the right fluor for the job!

i.e. pick a bright fluor for a dim epitope

and avoid spillover of brightcell populations into detectorchannels that require highsensitivity for rare signals

Problems in Emission Fluorescence

Spectral overlap

Excitation Emission

Optical solutions to spectral overlap: Filters

Filters resolve overlapping wavelengths of emitted light

Longpass filter: transmits light of longer than or equal toa specific wavelength

Shortpass filter: transmits light of shorter than or equal toa specific wavelength

Bandpass filter: transmits light only within a narrow rangeof wavelengths

Examples of optical filters in flow cytometry

Optical detector configurations

octagon

660/20

APC

735 LP

780/60

APC-Cy7

red trigon

bandpass

bandpasslongpass

EMISSION

two bandpass filters

Electronic solutions to spectral overlap: Compensation

To correct for emission spillover of FITC signal (normally detected in the FL1 channel) into the FL2 channel (which detects PE), it is necessary to use filters or electronic compensation or both.

Uncompensated Optimal

COMPENSATION

Before After

Multicolor immunophenotyping

No antibody. Autofluorescence only. No compensation applied.

CD4-PE. No compensation applied.

CD4-PE. Correct compensation applied.

1.4% PE subtracted from FITC PMT, 6.5% PE subtracted from APC PMT.

CD8-FITC. No compensation applied.

CD8-FITC. Correct compensation applied.

12.5% FITC subtracted from PE PMT.

CD4-PE + CD8-FITC. Streptavidin-APC alone.

CD4-PE + CD8-FITC. CD3-biotin + Streptavidin-APC

3 COLORS, CORRECTLY COMPENSATED

Spectral overlap of some fluorochrome combinationscannot be compensated easily or at all

Cy5APC

Therefore, avoid such combinations

Contour plots provide more accurate data representation than dot plots

Gates

gra

nula

rity

→

size →

The uses of gates for cell sorting

FSC

SS

CC

D8

CD3

Four Applications

Multicolor immunophenotyping

Cell cycle analysis

Phosphoprotein and kinase signaling

Stem cell sorting by the “side population” method

Cell cycle analysis

7-aminoactinomycin D

an

ti-B

rdU

-FIT

C

(DN

A

syn

thesi

s)

DNA content (propidium iodide)

G0/G1

G2/M SG0/G1

G2/M

S

Linear detection

2N 2N 4N4N

Multicolor, Auto Compensation with Flow Jo

100 101 102 103 104

FITC

0

20

40

60

80

100

% o

f M

ax

100 101 102 103 104

PE

0

20

40

60

80

100

% o

f M

ax

100 101 102 103 104

Pacific Blue

0

20

40

60

80

100

% o

f M

ax

100 101 102 103 104

PE-Cy7

0

20

40

60

80

100

% o

f M

ax

isotype isotype

isotypeisotype

0 200 400 600 800 1000

100

101

102

103

104

gate 1:lymphocytes

gate 2:B cells

gates 3 – 5

up to 11 colors

gate 6:T cells?

etc

Filter configurations permit optimizationof multicolor stains

When two colors are not enough

A single PBMC sample simultaneously stained with antibodies to quantify expression of CD3, CD4, CD8, CD7, CD27, CD28, CD45RA, CD62Land CCR7.

A lymphocyte size gate and CD3+/CD4-/CD8+ color gate is appliedto characterize stages of T celldifferentiation.

The T cell compartment cannot be fully characterized by only 2 or 3 markers.

Current Protocols in Immunology 12:12 (2005)

Other markers

FluorochromeMarker

B220: B lineage

IgM: mature B cells

IgD: mature B cells

CD23: transitional B cells

AA4.1: transitional B cells

Pacific Blue

PE-Cy7

PE

FITC

APC

CD45.1: donor phenotype

CD11b/Mac-1: myeloid lineage

live/dead discrimination*

CD45.2-Biotin: recipient phenotype

CD3: T lineage

PerCP-Cy5.5

APC-Cy7

Invitrogen UV-activated vital dye

Streptavidin Alexa 350

Qdot 605

Example of a 10-color experiment on our LSR II

FITC

FITC

FITC Compensation Matrix

“Phosphoflow” techniques allow you to measure kinase cascades and signal transduction

CourtesyJohn Meyers

johnm@bu.edu

Hoechst red

Hoech

st

blu

e

0 64 128 192 256FL4

0

64

128

192

256

FL5

0 64 128 192 256FL4

0

64

128

192

256

FL5

0.25%

Control Verapamil

Identification of a Hoechst 33342-staining ‘side population’ from murine bone marrow

100% of the gated side population is also Sca1+ ; these are hematopoietic stem cells.

Kinetic identification of “side population” stem cells

These gated stem cells can be isolated by MoFlo

FCCF rates

http://www.bu.edu/cores/flow-cytometry

On-line schedulingActual cytometer and work station use tracked to the minute,

recorded in and billed monthly by enterprise class central server

EquipmentUnassisted

rate (per hour)Assisted rate

(per hour)MoFlo NA $80.00LSR II $45.00 $67.50

FACScans $40.00 $67.50FACScalibur $40.00 $67.50Workstations $10.00 NA

Help and training:

Please sign up for basic training on FACScan or LSR2

with Yan Deng (X4-5225), ydeng@bu.edu

Problems during experiments:

We can’t read minds. Please write a computer entry

in the COMPLAINT LOG for each instrument. Or email us: flowcore@bu.edu

We will contact you ASAP and usually can respond within 2 hours.