Basics of Flow Cytometry

Holden Maecker

Outline

Definitions, what can be measured by flow cytometry

Fluidics: Sheath and sample streams, flow cells, sorting

Optics: Lasers, filters Electronics: PMTs, signal processing Fluorochromes: spectra, spillover Data analysis: FSC files, gating, statistics

Definitions

Flow cytometry: study of cells as they move in fluid suspension, allowing multiple measurements to be made per cell.

FACS™: fluorescence-activated cell sorting

What measurements can be made?

Forward light scatter (FSC): proportional to cell size

Side light scatter (SSC): proportional to cell granularity

Fluorescence: Binding of fluorescent-labeled antibodies Ca++-sensitive dyes within cells Fluorescent proteins expressed by cells Binding of DNA dyes

Scatter profile of lysed whole blood

Sid

e S

catt

er

Forward Light Scatter0 200 400 600 800 1000

02

00

40

06

00

80

01

00

0

Lymphocytes

Monocytes

Granulocytes

largest and most granular population

smallest and least granular population

Fluorescence data display

PE

FITCFITC Fluorescent Intensity

Num

ber

of E

vent

s Negative control histogram

Major components of a flow cytometer

Sample intake port Sheath and waste reservoirs Flow cell

Laser(s) Optical filters

PMTs (photomultiplier tubes) or photodiodes Signal processor

Cytometer fluidics create laminar flow

Sample stream

Sheath stream

Cell

Laser beam

Flow Cell

Cell sorting

Typical 2-color cytometer configuration

530/30 nm band pass filter

488nm band pass filter

488nm laser beam

560nm short passdichroic mirror

585/42 nm band pass filter

FL2PMT

FL1PMT

FSC PDflow cell

1% ND frontsurface mirror

488/10 nm band pass filter

SSCPMT

Background and autofluorescence

All cells have a certain level of background fluorescence, due to: Autofluroescence: from pigments and fluorescent

moieties on cellular proteins Non-specifically bound antibodies, and free

antibody in the sample stream The level of autofluorescence varies with the

wavelength of excitation and collection: Highest in FITC, PE detectors; lowest in far red

(APC, Cy7) detectors

Fluorescence sensitivity

Detection Efficiency (Q): number of photoelectrons generated per molecule of fluorophore Dependent upon fluorophore, filters, PMT

sensitivity, voltage gain setting, etc. Background (B): non-specific signal intrinsic

to the system Dependent upon autofluorescence, unbound

fluorophore, stray light, etc.

Common fluorophores for Ab conjugation

FLUOROCHROME Type of molecule Typical excitation laser Approximate emission peak

Fluorescein isotyocyanate (FITC)

Small organic 488 nm 518 nm

AlexaFluor 488 Small organic 488 nm 518 nm

Phycoerythrin (PE) Protein 488 or 532 nm 574 nm

PE-Texas Red Protein tandem 488 or 532 nm 615 nm

PE-Cy5 Protein tandem 488 or 532 nm 665 nm

Peridinin chlorophyll protein (PerCP)

Protein 488 or 532 nm 676 nm

PerCP-Cy5.5 Protein tandem 488 or 532 nm 695 nm

PE-Cy7 Protein tandem 488 or 532 nm 776 nm

Allophycocyanin (APC) Protein 633 nm 659 nm

AlexaFluor 647 Small organic 633 nm 667 nm

AlexaFluor 700 Small organic 633 nm 718 nm

APC-Cy7 Protein tandem 633 nm 784 nm

Pacific Blue Small organic 405 nm 454 nm

AmCyan Protein 405 nm 487 nm

Fluorescence spillover

Emission of FITC in PE channel

Compensating for spillover

uncompensated compensated

FITC mean fluorescence PE mean fluorescence---------------------------- ----------------------------negative positive negative positive----------- ---------- ----------- ----------

uncompensated 125 3540 185 1650 compensated 125 3560 135 135

1650 - 185

3540 - 125% Spillover = X 100

FCS files

FCS 2.0 and FCS 3.0 conventions Often referred to as list-mode files Contain all of the measurements (FSC-H,

FSC-A, SSC-H, SSC-A, FL1-H…) for each individual cell processed in a given sample

Hierarchical gating

Web reference tools

BD Spectrum Viewer:http://www.bdbiosciences.com/spectra

Maecker lab weblog:http://maeckerlab.typepad.com

(protocols, manuscripts, literature updates)