If I wanted to look at mtorc1 activity with ps6 as a readout in cd4+ t cells from wt and mutant, I can just pool cells. I noticed that most protocols do not explicitly state whether or not the t cells were purified ( MACS negative isolate, for example) prior to fixation and subsequent staining with the barcoding dyes.
If you use multi-cell populations then specific cell populations must be gated for using size, side scatter or fluorescence of a unique antibody. Otherwise you will not be able to separate them.
If I understand your question correctly, enriching for specific population (CD4+) T cells with magnetic beads for example will help you to get rid of dead cells (which after fixation will give high background in your staining) and cell populations (granulocytes, monocytes, etc) with high background for any fluorochromes used. Lymphocytes are the easiest cells to deal with in flow cytometry due to their low spontaneous cell death and low autofluorescence. So there will be less need in fixable viability dyes to gate away dead/apoptotic cells in staining. On mixed populations you can use "lymphacytic gate" on forward vs side scatter plot, but after fixation it will be difficult to set up this gate as side and forward characteristics of T cells will change after fixation.
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