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Flowcytometry Protocol

From Givan, A.L. (2000), chapter in In Living Color: Protocols in Flow Cytometry and Cell Sorting (R. Diamond and S. DeMaggio, eds). Springer, Berlin, pp 142-164.

Cell number of flow cytometry:

For each sample, you will need between 10^5 and 10^6 cells. If you are new to flow cytometry, use the higher number of cells -- to give yourself a margin for error (you always lose more cells than you expect during the staining and washing procedures). Trying to stretch too few cells into too many sample tubes in order to maximize results from a single experiment almost always leads to no results at all (it’s happened to all of us).

After staining and washing, resuspend the cell pellet in 0.3-1.0 ml, according to the configuration of the sample uptake on the cytometer. Use the smallest volume you can; the suspension can always be diluted with buffer later if cells are flowing too fast. When at the cytometer, adjust this final concentration of cells with more buffer if necessary, so that cells flow through the cytometer at about 100-1000 cells per second. Slower than 100 cells per second gets very boring. Faster than 1000 cells per second may lead to problems because the computer may not keep up or because two cells close together in the laser beam may be seen by the cytometer as a single "event." Constraints on adjusting cell concentration come from the total number of cells that are available and the minimum volume that your cytometer can handle. In general, you want to have cells at a high concentration so that you can use the lowest pressure possible to push them through the cytometer (but still achieve a flow rate close to 1000 cells per second). Pushing the cells too hard to achieve this high flow rate has deleterious effects on intensity resolution (by widening the sample core in the flow stream).

If you are setting up a new procedure or are needing to adjust instrument settings, it is especially helpful to have a few extra control samples with more cells in a larger volume. Running these cells initially, before you start collecting data , will give you time to adjust FSC and SSC parameters so that all cells are on scale, to make sure that background fluorescence is at an appropriately low position on the fluorescence scale, and to check out compensation if necessary for multicolor analysis.