I'm doing some extensive phenotyping by FACS of human lymphocyte populations. I'm looking at a number of markers- 41BB CD137 OX40 CD134 PD1 CTLA4 TIM3.... For many of these markers the isotype (using the exact one stated by the manufacturer) does not seem to work - the issue seems high background/baseline staining in unstimulated cells... ie, the staining of unstimulated cells yields a histogram that is broader than the isotype. Based on the dot plots, it doesn't look like a discretely positive population as opposed to when I do stimulate with OKT3/CD28/IL2. Does anyone have any explanation for this? I am resolving to use unstimulated PBMC as a control, but it doesn't seem correct. I have tried increasing the Fc Block, increasing the volume of isotype and decreasing the volume of stain. thanks!
Daniel
I think FMO controls would be better than isotype controls in this situation, due to the number of markers you have & the numerous uncharacterized interactions between their fluorescence spectrums. Also, try using compensation beads rather than stained cells to set as your compensation controls; they help set gates when expression is a gradient as opposed to discrete positive & negatives.
Thanks for the input. I tried FMO and it was identical to the isotype, thin, narrow peak by histogram. to be clear, my gating is FSC/SSC; CD3+/PI-; CD3+/CD4+ (or CD 8 when i stain for CD8 separately); histogram for the marker (always on APC for these markers). Im doing each marker individually on a BD Canto II
Thanks for the input. I tried FMO and it was identical to the isotype, thin, narrow peak by histogram. to be clear, my gating is FSC/SSC; CD3+/PI-; CD3+/CD4+ (or CD 8 when i stain for CD8 separately); histogram for the marker (always on APC for these markers). Im doing each marker individually on a BD Canto II
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