FACS ANALYSIS - (Jul/04/2009 )

Can nyone tell me wats d minimum cell number required for doing FACS analysis.

the minimum I used was 5.10^4 cells, in 200 µL on a facscalibur , but for more security I would say that the minimum volume should be 300 µL. I was analysing 5000 cells.

Yea thanks for the suggestion.At present am processing with 10,000 cells (no idea how much will be left after i do all my washing steps).Finally i resuspend the cells in 500ul FACS buffer n take 4 analysis.But the total events am getting is very less.

it might depend on what U want to analyse.

If U r just looking for just presence or absence of some antigen in same kind of cells (like if mouse cells express antigen X), less might be ok but if you want to see composition of some sample (as for example % of DC in PBMC or something like that) then more the cells in the sample, better the result.

It depends on what you are looking for. If you analyzing a small population within a sample then you will need to collect more cells. For example, if you are analyzing markers on hematopoietic stem cells in bone marrow (under 1%), you may need to collect up to 5 million live events in order to get a good analysis. If you are analyzing markers on CD4 cells from a mouse spleen, 10-20,000 cells will be sufficient. Try to determine the percentage of your cells you expect to see in a sample and then determine how many events you will need to collect for your marker. For a histogram analysis, having at least 5,000 events in the gate will get you a good looking histogram.

Thank u so much.Am pursuing binding detection of a ligand on cell surface receptor (high affinity binding). in fsc and ssc graph different groups of population can b seen though its a single cell line ( absence of cross contamination of cell line confirmed).Am using a single cell line, so am not sure which region to gate. Gating and without gating shows a different % positive cells. which one should i rely upon?

I would say that you have dead or dying cells on the left. try a propidium iodide staining to see dead cells.

can u post the graph.

In addition to the Facs data, can you provide your protocol?