Sample
Requirements - Flow Cytometry
ANALYSIS
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Cells must be in suspension, either in PBS or tissue culture medium.
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Cell concentration should be from 0.5 to 1 x 10E6 cells/ml with a minimum
volume of 0.2 ml (minimum of 1 to 2x105 cells).
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It is important that you do a cell count on your sample before bringing
them to the FACS lab it is important that
you know how many cells you have.
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Cell samples should be in 6-ml, polypropylene, round bottom tubes (Falcon
#2063). Other brands of round
bottom tubes may appear to be the same as the Falcon 2063 tubes but they
are not (many have a smaller inner diameter). Be sure your samples are
in the correct tubes otherwise they won't fit on the FACS sample pickup
line and time will be wasted in transferring them to the proper tubes.
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Samples should be free of cell clumps and debris.
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Viability of cell sample should be high, preferably =/> 90%
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It is also important to include positive and negative controls if possible.
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A typical experiment might contain the following samples:
Negative controls
Cells without any added antibody (autofluorescence)
Cells plus a non-reactive antibody (non-specific binding control, also
called isotype control).
Positive control
Cells plus an antibody known to bind to the cells being used.
Experimental
Cells plus antibody in question (including perhaps several dilutions)
Other controls that are sometimes included
Cells that would not be expected to bind the experimental antibody being
tested. For example, if
the antibody being tested is reactive with malignant cells, you may want
to include a "normal" cell to
rule out non-specific binding.
SORTING
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Cells must be in suspension.
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Cells should be at a concentration of 5 to 10 x 10E6/ml. The total number
of cells required will depend on the
number of sorted (purified) cells you require the percentage represented
by the cells of interest.
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For example, if the cell you wish to sort represents 10% of the total cells
and you require 1 x 10E7 purified
(sorted) cells, you would need to bring 1 x 108 stained cells to the FACS
lab assuming 100% recovery, which is
never the case. For example,
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Typical Sort Recoveries on the FACSVantage
Flow Rate
(cells/sec) 1,000 2,000
5,000 10,000
Recovery (*)
95% 90%
70% 60%
Purity (**)
99% 99%
99% 99%
(*) the percentage of positive cells actually
recovered during the sort.
(**) the percentage of the recovered cells
that are positive for the marker under investigation [CD4 in the example
below].
Example of sort recovery.
A sample of 36 x 10E6 cells is brought to
the FACS lab after being stained with a CD4 antibody; FACS analysis
indicates the sample has 35% fluorescence-positive
(CD4+) cells in it. The sample requires 1 hr of sorting time (at
10,000 cells/sec). At 36% positivity, the
theoretical yield of CD4 cells would be 12 x 10E6 cells: [(36 x 10E6) x
(0.35)] At a recovery rate of 65%, we are
able to collect 8 x 10E6 cells [(12 x 10E6) x (0.65)] which will be =/>99%
pure, i.e., 99% of them will be CD4+.
