1. Introduction to the Principles of Flow Cytometry
for users of the
Flow Cytometry Core Facility
at BUMC
21 September 2010
Mike Xie (X4-5225),
Yan Deng (X4-5225),
John Meyers (X8-7543),
Gerald Denis (X4-1371),

2. 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

3. Where are the instruments located?
LSR II,
MoFlo:670-5
FACScan:
R901
X620
FACScan,
FACSCalibur:L508
U R here

5. Flow cytometry was not easy in the old days
ungloved hands
missing PPE
gendered division
of labor
live anthrax
bacilli

6. But today… it’s as easy as PCR.

7. 3 elements of any
flow cytometry system
fluidics
optics
electronics

8. fluidics

9. Fluidics sample fluid Purpose of the fluidics system:
sheath
fluid
Purpose of the fluidics system:
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
‘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.

10. 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’

11. optics
electronics
“SSC”
“FSC”

12. Forward and side scatter of leukemic cells
Normal B cells
Malignant, large B cells
SSC
FSC
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 to
size, shape and internal structure/irregularity of the cells.

13. Photomultiplier (PMT) detectors convert photons
(selected by mirrors and filters) to electrical pulses
Peak 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.

14. Photomultiplier Tubes (PMTs)
FSC: forward scatter
(size; cross-sectional
area)
SSC: side scatter
(granularity, internal
or surface structure
that scatters light)
Adjusting the voltage
of the PMT helps to
optimize capture
of desired populations

15. A dotplot represents two properties of a single cell
SSC
FSC

16. Control Condition Experimental
A histogram represents the distribution of a single parameter across many cells
10,000 cells each!
Control Condition Experimental
1 cell
1 cell
1 cell
1 cell

17. 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)
(Log detection)

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

19. 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!

20. FLUORESCENCE Excitation wavelength and emission wavelength are
blue laser
Excitation wavelength and
emission wavelength are
unique properties of
each specific molecular
structure
(FITC)

21. Stokes Fluorescence
Excitation
Emission
Stokes shift

22. yes no Laser light must overlap with excitation wavelength
Fluorescein (FITC)
Propidium iodide (PI)
488
488 ex em
yes ex em
Hoechst
Texas Red
488
488 no ex em ex em

23. Widely-used molecules are excited by the 488 nm laser (FACScan)
488 nm (Blue) : FITC, GFP, PE, PerCP, PE-Cy5, PI,
PerCP-Cy5.5, PE-Cy7
But different lasers are available to excite
other molecules (LSR II)
355 nm (UV) : Indo-1, DAPI, Alexa Fluor 350, Hoechst 33258
405 nm (Violet) : Alexa Fluor 430, Alexa Fluor 405, Pacific Blue
561 nm (Yellow/Green): Texas Red, Cherry Red, Tomato Red
633 nm (Red): APC, APC-Cy7, Alexa Fluor 647, Alexa 680

24. Octagon Detector Arrays
emitted fluorescent light
longpass dichroic mirrors
bandpass filters

25. EMISSION

26. Fluorescence detection
autofluorescence
weakly expressed
epitope
strongly expressed
epitope
Note logarithmic scale

27. Isotype controls weakly expressed epitope isotype control test
How do you know
it’s real?
weakly expressed
epitope
isotype control test
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.

28. Resolution sensitivity
Resolution sensitivity, the ability
to resolve a faint signal from
background) depends on the
difference D between the positive
and background peaks and the
spread of the background
peak W

29. Choose the right fluor for the job!
Reagent Stain Index
Phycoerythrin (PE)
Alexa Fluor
APC
PE-Cy
PE-Cy
PerCP-Cy
PE-Alexa Fluor
Alexa Fluor
FITC
PerCP
APC-Cy
Alexa Fluor
Pacific Blue
AmCyan
i.e. pick a bright fluor for a
dim epitope
and avoid spillover of bright
cell populations into detector
channels that require high
sensitivity for rare signals

30. Problems in Emission Fluorescence
Excitation
Emission
Spectral overlap

31. Optical solutions to spectral overlap: Filters
Filters resolve overlapping wavelengths of emitted light
Longpass filter: transmits light of longer than or equal to
a specific wavelength
Shortpass filter: transmits light of shorter than or equal to
Bandpass filter: transmits light only within a narrow range
of wavelengths

32. Examples of optical filters in flow cytometry

33. Optical detector configurations
octagon
red trigon
660/20
APC
735 LP
780/60
APC-Cy7
bandpass
longpass
bandpass

34. EMISSION
two bandpass filters

35. 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

36. COMPENSATION
Multicolor immunophenotyping
Before
After

37. No antibody. Autofluorescence only. No compensation applied.

38. CD4-PE. No compensation applied.

39. CD4-PE. Correct compensation applied.
1.4% PE subtracted from FITC PMT, 6.5% PE subtracted from APC PMT.

40. CD8-FITC. No compensation applied.

41. CD8-FITC. Correct compensation applied.
12.5% FITC subtracted from PE PMT.

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

43. CD4-PE + CD8-FITC. CD3-biotin + Streptavidin-APC
3 colors, correctly compensated

44. Therefore, avoid such combinations
Spectral overlap of some fluorochrome combinations
cannot be compensated easily or at all
APC
Cy5
Therefore, avoid such combinations

45. Contour plots provide more accurate data representation than dot plots

46. Gates
granularity →
size →

47. The uses of gates for cell sorting
SSC
FSC
CD8
CD3

48. Four Applications
Cell cycle analysis
Multicolor immunophenotyping
Phosphoprotein and kinase signaling
Stem cell sorting by the “side population” method

49. DNA content (propidium iodide)
Cell cycle analysis
G0/G1
G2/M S S
anti-BrdU-FITC
(DNA synthesis)
G2/M
G0/G1 2N 4N 2N 4N
DNA content (propidium iodide)
7-aminoactinomycin D
Linear detection

50. Multicolor, Auto Compensation with Flow Jo20 40 60 80
100
% of Max
isotype 20 40 60 80
100
% of Max
isotype
gate 1:
lymphocytes
gate 2:
B cells 10 1 2 3 4 10 1 2 3 4
gate 6:
T cells?
FITC
Pacific Blue 20 40 60 80
100
% of Max
isotype 20 40 60 80
100
% of Max
isotype
etc
200
400
600
800
1000 10 1 2 3 4
gates 3 – 5 10 1 2 3 4 10 1 2 3 4 PE
PE-Cy7
up to 11 colors

51. Filter configurations permit optimization
of multicolor stains

52. 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, CD62L
and CCR7.
A lymphocyte size gate and
CD3+/CD4-/CD8+ color gate is applied
to characterize stages of T cell
differentiation.
The T cell compartment cannot be
fully characterized by only 2 or 3
markers.
Current Protocols in Immunology 12:12 (2005)

53. Example of a 10-color experiment on our LSR II
Other markers
Fluorochrome
Marker
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

54. FITC Compensation
Matrix
FITC
FITC

55. “Phosphoflow” techniques allow you to measure
kinase cascades and signal transduction
Courtesy
John Meyers

56. Identification of a Hoechst 33342-staining
‘side population’ from murine bone marrow
Hoechst red
Hoechst blue
0.25%
Control
Verapamil
100% of the gated side population is also Sca1+ ;
these are hematopoietic stem cells.

57. Kinetic identification of “side population” stem cells
These gated stem cells can be isolated by MoFlo

58. FCCF rates http://www.bu.edu/cores/flow-cytometry On-line scheduling
Actual cytometer and work station use tracked to the minute, recorded in and billed monthly by enterprise class central server

59. Problems during experiments:
Help and training:
Please sign up for basic training on FACScan or LSR2
with Yan Deng (X4-5225),
Problems during experiments:
We can’t read minds. Please write a computer entry
in the COMPLAINT LOG for each instrument.
Or us:
We will contact you ASAP and usually can respond within 2 hours.