This document provides an introduction to flow cytometry. It defines flow cytometry as the measurement of physical and chemical characteristics of cells as they flow in a fluid stream through a beam of light. It describes the key components of a flow cytometer including fluidics to deliver cells to the laser, optics to excite and collect light, and electronics to amplify and process signals. It explains the different types of signals detected including light scatter and fluorescence, and how these can be used to characterize cells. The document provides guidance on choosing fluorochromes and considerations for multi-color panels such as spectral overlap. It outlines some common applications of flow cytometry and contact details.

1. MLC Flow Cytometry Facility
Introduction To Flow
Cytometry
Rob Salomon
Garvan Institute of Medical Research
Darlinghurst NSW
Flow Cytometry

2. What Is Flow Cytometry ?

3. What Is Flow Cytometry ?
Measurement
METRY

4. What Is Flow Cytometry ?
Cells Measurement
CYTO METRY

5. What Is Flow Cytometry ?
Flow Cells Measurement
Flow CYTO METRY

6. What Is Flow Cytometry ?
Flow Cells Measurement
Flow CYTO METRY
Flow Cytometry

7. Prerequisites for Flow
Cytometry
1. Cells in single cell suspension
2. Fluorescent probes
3. Cytometer
The key to good
result is good
sample
Preparation
http://www.photobiology.info/Zimmer.html -
from Roger Y.Tsien)

8. What does a Flow Cytometer
do?
Analyses light signals to determine:
Phenotype and Function
Cd3

9. What’s inside a Flow
Cytometer ?
• Flow cytometers have 3 key systems
– Fluidics
– Optics
– Electronics

10. Fluidics
Delivery of
Low
sample to
laser
intercept
(interrogation
Medium point)
Legend
Laser intercept
High Core Stream

11. Optics
• Allow the excitation and the collection of
the emitted light
Steering
LASER mirrors
emission
Flow Cell -
Steering interrogation
mirrors
point

12. Optics cont..
Fluorescent
and SSC
Detectors
Signal Detection
FSC is achieved by
detector collecting emitted
or scattered light

13. Optics cont..
B530 Detector
– FITC GFP
530/30
488/10
SSC
Fluorescent and
Detector
506 LP SSC signals are
collected at right
Emission angles to the
from blue 575/26
excitation laser
laser are progressively
B575 Detector
picked off to
– PE, PI
556 LP
facilitate multiple
fluorochrome use

14. Electronics
Detector or PMT
Electron Cascade
Digitisation
and
processing
Amplification Voltage
http://sales.hamamatsu.com
/assets/applications/ETD/p
mt_handbook_complete.pdf

15. What type of signals do we
see with Flow ?
• Scatter
– Forward Scatter (FSC)
• parallel or Perpendicular FSC
– Side scatter (SSC)
• Fluorescence
– FITC , PE, APC, GFP, DAPI (plus lots lots more)

16. Understanding Scatter Signals
• WBC discrimination
FSC has some
similarities to size
SSC has some
similarities to
granularity and
complexity

17. Fluorescent Signals
• Fluorescence may be used in the
detection of :
– Protein, RNA and DNA
– DNA synthesis
– Dye efflux
– Organelle Activity
A cytometer can
– Change in pH detected light from
– Protein interactions any system you
can design that
– Cell movement and division
utilises
– etc fluorescence

18. Examples of fluorescent probe
use 10
5
Fluorescence 2 (CD4)
<B670L_B-A>: CD8-PerCP55
104
3
10
0
0 103 104 105
<R780_A-A>: bTCR-APCAF750
Fluorescence 1 (βTCR)

19. Understanding Fluoroscence
The fluorescent
Excited
molecule is excited
e-
by the excitation
state
e- source (laser). This
imparts energy to
e-
electrons in the
e-
molecule which in
Resting e-
then released as
Mechanism of the molecule
relaxes. The
Fluorscence energy is released
as light.

20. How do I choose my
Fluorochromes ?
• Antibody availability
• Function – i.e. Mcherry Vs GFP
• Fluorochrome brightness
• Excitation source
• Emission filters
• Other fluorochromes/ Signals present in my
sample (spectral overlap)

21. Fluorochrome Brightness
Probe QY
AF488 0.92
R-Pe 0.82
AF546 0.79
AF594 0.66
Quantum yield :
APC 0.68
Is a measure of the
A647 0.33 relative brightness of
eGFP 0.6 the fluorochrome. IT
is measured as:
Azumi Green 0.74
ZS Green 1 0.91
http://en.wikipedia.org/wiki/Fluorophore

22. Fluorescent protein table
http://www.tsienlab.ucsd.edu/Publications/Shaner%202005%20Nature%20Method
s%20-%20Choosing%20fluorescent%20proteins.pdf

23. Choosing your Fluorochromes
spectral
viewers
http://www.bdbioscience
s.com/research/multicolo
r/spectrum_viewer/index.
jsp
http://www.invitrogen.co
m/site/us/en/home/supp
ort/Research-
Tools/Fluorescence-
SpectraViewer.htmlUse
the

24. Choosing your Fluorochromes
spectral
viewers
http://www.bdbioscience
s.com/research/multicolo
r/spectrum_viewer/index.
jsp
http://www.invitrogen.co
m/site/us/en/home/supp
ort/Research-
Tools/Fluorescence-
SpectraViewer.htmlUse
the

25. Choosing your Fluorochromes
spectral
viewers
http://www.bdbioscience
s.com/research/multicolo
r/spectrum_viewer/index.
jsp
http://www.invitrogen.co
m/site/us/en/home/supp
ort/Research-
Tools/Fluorescence-
SpectraViewer.htmlUse
the

26. Choosing your Fluorochromes
spectral
viewers
http://www.bdbioscience
s.com/research/multicolo
r/spectrum_viewer/index.
jsp
http://www.invitrogen.co
m/site/us/en/home/supp
ort/Research-
Tools/Fluorescence-
SpectraViewer.htmlUse
the

27. Understanding Spectral
Overlap
Effect of spectral overlap - Instrument View
120%
100%
Percentage of Signal
80%
in Detector
60%
40%
20%
Spectral overlap
0%
B 530 B 585 occurs when
PE 5% 87% fluorochromes
FITC 95% 13%
excited by the
same lasers emit
in similar ranges.

28. Compensation
Signal from FITC bright
Compensation Controls 120
Signal Strength
100
120% 80
60
100% 40
20
80%
Axis Title
0
overlap B 530 B 585
60%
FITC bright 100 13
40% overlap
20% FITC dull
0% 120
Signal Strength
B 530 B 585 100
FITC 100% 13% 80 Compensation is
PE 5% 100% 60
40 applied at the
20
0 single event
B 530 B 585
FITC dull 50 6 level

29. Effect of Compensation
Digital
compensation
doesn’t change the
underlying data it
just allows us to
Uncompensated Data interpret it

30. Effect of Compensation
Digital
compensation
doesn’t change the
underlying data it
just allows us to
Compensated Data interpret it

31. How many Fluorochromes can
I use ?
• Most flow = 1- 3 fluorochromes
• Basic phenotyping panel = 6-8
fluorochromes
• Complicated panels = 11-12
flourochromes
• High end = 17 fluorochromes
Seventeen-colour flow cytometry: unravelling the immune system
Stephen P. Perfetto, Pratip K. Chattopadhyay & Mario Roederer

32. Impact of increasing Flourochromes
• Data get dramatically more complex
Parameters 2 3 4 8 12 18 22
Populations 22 23 24 28 212 218 222
Populations 4 8 16 256 4,096 262,144 4,194,304
With 3 12 24 48 768 12,288 786,432 12,582,912
scatter
populations
Number of populations – assuming each fluorochromes gives
rise to only a positive and negative population

33. Visualising Signal Data
Dot Density
plot plot
Statistical
measures are
also used to
Contour
plot identify
Histogram
changes

34. Basics uses of Flow
Cytometry ?
• Phenotyping
• Apoptosis and cell death
• Cell cycle, cell divising and DNA synthesis
• Transduction/transfection confirmation
• Cell tracking
• Small particle analysis
• Functional analysis – calcium flux, gene expression, dye
efflux, mitochondrial activity
• Marine and microorganism identification

35. Why use Flow Cytometry ?
• Rapid analysis ( 3k- 200k events/second)
• Individual event analysis
• Quantifiable results
• Multiple parameter analysis
• Statistical relevance

36. Flow and Imaging Comparison
Imaging Flow Cytometry
Cells per field/sec) Approx 100 20, 000
No. of parameter <6 <24
Quantifiable Maybe (using complex Yes
analysis tool) 18 bit resolution
12- 16 bit (< 65,536 (262,144 channels)
channels)
Ave number of 1,000 - >10, 000
analysed cells 10 field of 100
Anatomical localisation Yes no

37. How do I get more ?
Analysis
Cell Sorting
Sorting
See It
Sort It

38. Contact Details
• Rob Salomon
– r.salomon@garvan.org.au
– (02) 9295 8431
• Bookings (Nikki and David)
– Flow@garvan.org.au
– (02) 9295 8432
• http://linkage.garvan.unsw.edu.au/Flow/index.html