Basics of Flow Cytometry

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Basics of Flow Cytometry

• Holden Maecker

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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

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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

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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

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Scatter profile of lysed whole blood
Side Scatter
Forward Light Scatter
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Fluorescence data display
Negative control histogram
PE
Number of Events
FITC Fluorescent Intensity ?
FITC
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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

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Cytometer fluidics create laminar flow
Sample stream
Flow Cell
Sheath stream
Laser beam
Cell
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Cell sorting
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Typical 2-color cytometer configuration
FL1 PMT

488/10 nm band pass filter
530/30 nm band pass filter

SSC PMT
1 ND front surface mirror

FL2 PMT
560nm short pass dichroic mirror
585/42 nm band pass filter
488nm band pass filter
488nm laser beam
FSC PD
flow cell
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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
• Generally higher in blue-excited channels than
  red-excited channels

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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.

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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
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Fluorescence spillover
Emission of FITC in PE channel
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Fluorescence spillover
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Compensating for spillover
uncompensated
compensated
FITC mean fluorescence PE mean
fluorescence ---------------------------- ------
---------------------- negative positive negativ
e positive ----------- ---------- ----------- --
-------- uncompensated 125 3540 185 165
0 compensated 125 3560 135 135
Spillover
X 100
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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

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Hierarchical gating
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Statistics and reporting of data

• Rare event assays (e.g., ICS) require larger
  numbers of events to be collected per file for
  statistical accuracy.
• Results are usually reported as a percentage of
  CD3CD4 or CD3CD8 lymphocytes.
• Conversion of these numbers into absolute counts
  per mL of blood may be done by multiplying by
  1000 x CD4 or CD8 count
• This may be advisable in patients with HIV or
  other conditions that alter T cell counts

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Acknowledgements

• Joe Trotter
• Spectrum Viewer
• http//www.bdbiosciences.com/spectra
• Laurel Nomura
• Protocols, manuscripts, literature updates
• http//www.maeckerlab.org