Download Running A Flow Cytometry Facility

Introduction to Flow
Cytometry
Shonna Johnston
Centre for Inflammation Research
University of Edinburgh
What is Flow Cytometry
• It is the technique used to measure properties of
individual cells as they flow in a stream one by
one past a sensing point
• This ability to measure cellular parameters based
on light scatter and fluorescence and to physically
purify subpopulations has led to widespread use of
this instrumentation in biology and medicine
Main Components of Cytometer
• Fluidics
• Optics
• Electronics
Fluidics
LASER
SHEATH
FILTER
FLOW
CELL
WASTE
SAMPLE
Hydrodynamic Focusing
FLOW CELL
488nm LASER
SAMPLE
Fluidics Affect of sample pressure differences
Sheath Pressure is constant at 4.5psi
LO
MED
HI
4.6psi
4.8psi
5.0psi
Sample Pressure
Note: Sample pressure is always higher than Sheath pressure to allow the sample to enter the flow.
Optics
Forward Scatter / Side Scatter
Refracted 488nm light
Small cell
FSC diode
detector
488nm Blue Laser
Large cell
Blocker
bar
Prevents the
laser light
blinding the
detector
488/10 BP
Allows light between 483-493
IE the refracted laser light
This diagram shows how a larger cell size gives a higher forward scatter reading. However
remember that this can be affected by the orientation of cells past the laser eg elongated shape
of some epithelial cells and any surface variations ie dendrites
Optics
FACSCalibur
FL-1
530/30 BP (515-545)
SSC
90/10 Beam Splitter
488/10 BP (483-493)
FL-2
DM 560 SP
585/42 BP (564-606)
FL-4
DM 640 LP
661/16 BP (653-669)
Half Mirror
670 LP
FL-3
Fluorescence collection lens
488nm Laser Blue
635nm Laser Red
(only used with FL4)
Flow
Cell
FSC
488/10 BP
Electronics
• The light falling on the photodetector surface generates a
current which produces a pulse
• This pulse can be processed into height, width and area
• Normally the height is displayed on the plots although it
is becoming more common to use area
•The electronics also allow the adjustment of spectral
overlap by compensation equations
Electronics
488nm
Spectral Overlap
FL1
FL2
Compensation
Compensation
Flow Sorting
• Ability to physically separate cells with different
properties
•Uses a stream-in-air flow which allows droplet formation
• Cells are found in droplets which can then be charged
• By using oppositely charged plates the drops are pulled to
the side into a collection tube
Cells
• Primary Cells
• lymphocytes, neutrophils, eosinophils, monocytes,
dendritic cells, platelets
• Cell Lines
• Transfectants (e.g. CHO, COS7)
• Leukaemic cell lines (e.g. MUTU, U937, THP1)
• Bacteria
• E. coli 0157
Applications
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Phenotyping
Apoptosis studies
Phagocytosis/Binding Studies
Cell Cycle/Kinetics
Rare populations - CD34+, side population
Intracellular cytokines
Intracellular calcium
Multiplex cytometric bead assays
Links / Further Reading
• http://www.cyto.purdue.edu
E-mail Forum for cytometry question
Lecture presentations
• http://flowcyt.salk.edu
Links to all major flow lab sites
• Practical Flow Cytometry (2004)
Howard Shapiro
•Flow Cytometry; A Practical Approach (2000)
Michael Ormerod