FLOW CYTOMETRY
I had dinner with my friend Celine and mentioned that I spent
my day on the FACS machine.
She was really surprised that a professor at McGill can spend an
entire day sending FAX to people!
I had to explain that FACS is not FAX but stands for fluorescenceactivated cell sorting or flow cytometry.
What is flow cytometry? It has something to do with cells?
Tell me more about it.
Let see if I did a good job in explaining to my friend Celine, who
has a good scientific background, what is flow cytometry and
whether you could do better!
Celine’s Questions
What is flow cytometry?
How does it work?
What are the basic components of a flow cytometer?
How analysis of individual cells is achieved?
Which type of information can you obtained from this technology?
How cell size is measured?
How cell granularity/complexity is measured?
How expression of various molecules by the cells is measured?
Give me an example of data for the expression of a given
molecule.
Which conclusions can you make from these data?
Is it possible to detect more than one molecule?
What is compensation?
How compensation is set?
What are the consequences of bad compensation?
Bold red questions = in-class problem
What is Flow Cytometry?
Flow cytometry….
How does it work?
individual cells in a fluid medium pass throug a laser beam and the light that emerges from
the cell is captured and transformed into quatititative data
What are the basic components
of a flow cytometer?
the fluidifc system,
which carried and
delivers indicial cells to
the lught source
the laserss which is
the light source
How analysis of individual cells
is achieved?
This is accomplished by hydrodynamic focusing in which cells in liquid
suspension are injected into a flowing stream of sheath fluid that is
compressed, forcing the cell into the center of the stream that has roughly
one cell in diameter.
Which type of information can you
obtained from this technology?
• Cell size
• Cell granulocyty/complexity
• Expression of several different cell surface
and/or intracellular molecules
How cell size is measured?
As a cell passes through the laser beam it will diffract or scatter
light in all direction. It is possible to measure the amount of light
scattered in the forward direction as a cell passes through the
laser beam.
Small cells scatter a small amount of light in the forward
direction while larger cells scatter a larger amount of light. The
amount of light scattered in the forward direction (the forward
scatter or FSC) is thus roughly proportional to the size of the cell.
How cell size is measured?
Relative cell number
The amount of forward scattered light is quantified by a detector that converts
light intensity into an electronic signal that is digitized and sent to a computer.
Small cells produce a small FSC signal and larger cells generate a higher FSC signal.
If a graph is plotted for the intensity of the FSC signal for each cell versus the
relative cell number in a sample, small cells appear on the left of the FSC axis
while larger cells appear further on the right.
It is thus possible to have a graphical representation of the size of the cells in a
given sample (this is called an histogram).
Cells from 0.2 to 50 um can be easily detected by flow cytometry.
FSC
FSC
How cell granularity/complexity is
measured?
Light scattering to the side (90°), is caused by granularity and complexity
inside the cell. This side-scattered light (SSC) is focused through a lens system
and is collected by a separate detector.
Two-dimensional representations
It is possible to have a graphical 2-D representation of combined parameters
for each cell. One example of such 2-D representation is called a dot plot.
Here is an example from human blood (in which red blood cells have been
partially lysed)
A two-dimensional diagram where the SSC signals are plotted against FSC
signals for individual cells reveals the presence of 3 cell types (each dot
corresponds to a cell):
Lymphocytes which are small cells possessing low internal complexity;
monocytes which are medium-sized cells with slightly more internal
complexity, and granulocytes which are large cells that have a lot of internal
complexity. The cells in the red gate are aggregated red blood cells and cell
debris.
4000
SSC
3000
2000
1000
0
0
1000
2000
FSC
3000
4000
How expression of a given molecule
by the cells is measured?
Cell surface and/or intracellular molecules are detected by
specific Abs labeled with fluorescent dyes (fluorophores).
The cells are labeled with these Abs and when they pass through
the laser beam the dye molecules bound to the cells are excited
and emit fluorescence which is detected by photomultiplier
tubes.
The amount of fluorescence emission of a given dye is
proportional to the number of labeled Ab bound to the cell.
Thus, fluorescence emissions give information on the binding of
the labeled Abs and hence on the expression of proteins by each
cell.
Give me an example of data for the
expression of a given molecule
Cells expressing low levels of the molecule produce a small fluorescent signal and cells
expressing higher levels of the molecule generate a higher fluorescent signal. If a graph is
plotted for the intensity of the fluorescent signal for each cell versus the relative cell
number in a sample (an histogram), low expressing cells appear on the left of the
fluorescent axis ( x axis) while higher expressing cells appear further on the right.
M1
Unstained cells
(autofluorescence)
M1
M2
100
101
102
iso gran b
103
104
100
101
M2
102
gran b
103
M1
104
100
population is more on the right in the first 2 graphs
M1 is the region of negaative
M2 is the regions of positivity
- in the 2 histograms, the one at the right produces less fluorescnece (the peak in M2 is lower
101
M2
102
Perforin
103
104
Which conclusions can you make from these data?
0
M1
200 400 600 800 1000
FSC
Based on the histogram of the unstained cells, I can put markers (M2) and
determine the percentage of cells that express the molecule of interest.
I can do this on different populations. Based on the SSC/FSC profile of my
sample I can choose to analyze a specific population (gated population) for
the expression of the molecule (red or blue population).
I can determine the mean expression level of the molecule between
different populations (red versus blue population).
Unstained cells
(autofluorescence)
M1
M2
100
File: 150208.005
Marker
All
M1
M2
fluorescence by the
population in the
blue circle
- larger more
complex cells —
less expression of
molecule
101
102
iso gran b
Events % Gated
7950
100.00
7874
99.04
92
1.16
103
100
104
101
M2
102
gran b
Mean Geo Mean Median
5.16
4.79
4.53
5.03
4.73
4.53
17.43
16.44
14.86
103
M1
104
100
File: 150208.004
Marker
All
M1
M2
Events % Gated
8010
100.00
50
0.62
7965
99.44
102
Perforin
Events % Gated
8032
100.00
1490
18.55
6676
83.12
File: 150208.006
Marker
All
M1
M2
101
M2
Mean Geo Mean Median
83.28
59.83
50.48
10.13
9.79
10.75
83.69
60.46
50.48
103
104
Mean Geo Mean Median
25.46
20.91
19.81
9.49
9.10
10.00
28.76
24.91
22.88
Can you draw a 2D representation of these data?
100
101
102
Foxp3
103
104
Is it possible to detect more than one molecule?
It is now possible to perform 21-color analysis (meaning that it is possible
to use the expression of 21 different cell- surface and/or intracellular
proteins to characterize various cell subsets in a given sample). Flow
cytometry is a very powerful tool.
When using more than one fluorophore, it is critical to compensate.
What is compensation?
What is compensation?
What is compensation?
What is compensation?
How to set compensation?
As an example we will perform a stain of lymph nodes cells for CD8 and CD4
T cells, using a CD4-PE mAb and a CD8-FITC mAb.
In addition to the experimental stain (cells + anti-CD4-PE + anti-CD8-FITC),
cells are also stained with individual fluorophores (cells + anti-CD4-PE only
and cells + anti-CD8-FITC only). Unstained cells are also necessary.
Unstained cells allow for setting forward scatter and side scatter voltages so
that the cell population is clearly delineated.
Unstained cells are also used to set the voltages for fluorescence channels in
such a way that the entirely unstained population is completely off
the lower axis for every parameter being measured.
(i.e. all the cells should appear in the lower left corner of the dot plot CD4 vs
CD8)
Cells stained with only the CD8-FITC mAb are
examined.
The bright FITC positive cells show an
appreciable signal in the FL2 detector.
The FL2-%FL1 compensation control is then
gradually increased until the median FL2
channel number of the FITC-bright cells is the
same as the median FL2 channel number of
the FITC-neg cells (the centers of the two
populations are equal).
Compensation is correctly set when the
median of the negative population is equal to
the median of the positive population in the
spillover channel. For example, if using a
positive FITC-stained sample to apply
compensation to the PE channel, the median
of the negative and the FITC-positive
populations should be equal in the PE channel.
Repeat for all channels into which FITC leaks (if
doing more than 2 color analysis).
Repeat for all single color controls.
Cells stained with only the CD4-PE mAb are
examined.
The bright PE positive cells show a signal in the
FL1 detector.
The FL1%FL2 compensation control is then
gradually increased until the median FL1
channel number of the PE bright cells is the
same as the median FL1 channel number of
the PE-neg cells (the centers of the two
populations are equal).
Compensation is correctly set when the
median of the negative population is equal to
the median of the positive population in the
spillover channel. For example, if using a
positive PE-stained sample to apply
compensation to the FITC channel, the median
of the negative and the PE-positive
populations should be equal in the FITC
channel.
Repeat for all channels into which PE leaks if
doing more than 2 color analysis.
What are the consequences of bad compensation?
Correctly compensated
Under compensated
Over compensated