Hi All,
I am trying to access changes in B cell markers in our knockout mouse and wild type from blood. I try to gate lymphocytes, then single cell, followed by live dead and B220+ cells. When i paste the same gate across all samples there seems to be a shift in population and also i can see different pattern across samples. Lot of dots towards lower FSC. I am attaching a file for reference. As we always take percentage, how can i then compare such population???
My suggestions are.
1. you can use miltenyi dead cell removal kit to remove dead cells, which will easen the problem of dead cells.
2. you should also standardize the flurochormes and voltages. I dont see a clear separation of the populations and the voltage probably is also too high.
3. you can further increase FSC and SSC voltages too.
4. when you carry over gates,most of the times it is better to have similar FSC and SCC voltages.
I would switch your gating strategy and you might need to adjust your settings.
Do the live dead gate first. Do the gate with the Zombie dye and the SSC. Gate on the negative (live) cells. (Just to double check that your Zombie dye was stained in PBS and not BSA? It's just that you should have a much better separation between live and dead cells. And besides, you shouldn't have that many dead cells...)
From the live gate, run the single cell gate. You can run it vice versa ( always do the live/dead gate first... clears things up for later). But these two first.
THEN you gate on the lymphocytes with the FSC/SSC. (BTW... I don't think that left lower cluster are lymphocytes. And I also don't see a clear neutrophil separation. There might be a monocyte one...but the visually, the numbers seems off.)
Then the B220.
The setting on your flow does seem off.
BTW...did you lyse the RBCs, or did you do a no lyse no wash protocol?