I am going to do mouse tumor phenotyping by flow. before going to tumor, I tested in mouse spleen to see how the fluorescence work and I only stained single fluorescence.

CD3,4 and 8 are always good., but CD44-PerCP and FOXP3-PE no positive group. I am wondering if this is because the expression level is low in mouse spleen (also could be antibody problem).

Does anybody use beads for compensation in mouse tumor immunophenotyping?

Or anybody has some experiences about the CD44 level and foxp3 level in spleen of normal black mouse? what material you use for compensation in mice?

Thanks very much!

the procedure of my assay is:

1 digested spleen to single cells---filter---wash twice---RBC lysis---wash twice--did cell counting

2 stain surface markers, fix with fixing and permeabilization kit. keep cells in permeabilizaiton buffer, 48 hours later went to flow machine

3 for foxp3 staining, fix and permeabilized using kit, stain, wash with PER buffer, keep in PER buffer 48 hours later went to flow machine

Thanks!