Flow Cytometer Set-Up

Basic Guidelines:

Adjust your flow cytometer settings by analysing a sample with unstained cells of the same type and given the same treatment as the cells in the rest of the samples that are to be analyzed.
Adjust your FS and SS settings so that all cell populations are visible. Normally the lymphocyte population is placed centrally in FS and low in SS.
Gate on your lymphocyte population (se figures below).
Adjust PMT settings for relevant channels to baseline level.
Flow cytometer is now ready for sample analysis. We recommend inclusion of viability staining. Gate away dead cells if possible, as they tend to blur the staining picture.

Gating:

	Gating on Mouse Splenocytes Splenocytes isolated from mouse analyzed on the CyAn™ Flow Cytometer. 10,000 events were collected. Two distinct populations appear. The population to the left contains a lot of dead cells. When staining splenocytes with MHC Dextramers, always gate on the population to the right because dead cells can obscure the picture.
	Gating on Ficoll Purified Human Lymphocytes Ficoll purified human peripheral blood mononuclear cells analyzed on the CyAn™ ADP Flow Cytometer. 125,000 cells were collected. Gate is shown around lymphocytes. Population marked 1 is monocytes. Thrombocytes and cell debris appear in the lower left corner. When staining with MHC Dextramers, gating on lymphocytes is recommended.
	Gating on Whole Blood Human whole blood was analyzed on the CyAn™ Flow Cytometer. 35,000 events were collected. Gate is shown around lymphocytes. Population marked 1 is granulocytes, population marked 2 is monocytes, and cell debris appears in the lower left corner. When staining with MHC Dextramers, it is recommended to gate for lymphocytes.