Hi everyone!
I want to study the response of plasma cells to different drugs after in vitro exposure. Can I culture whole bone marrow (after RBC lysis), treat with my drug, wait an appropriate amount of time, and use flow cytometry to analyze the plasma cell response? I'm using Annexin V vs 7-AAD for death analysis and surface marker expression for cell identity.
I'm asking because I've always used live/dead stains to exclude dead cells since they can bind antibodies nonspecifically. How can I trust surface staining to identify my cell of interest if a good portion of them are dead? Can I still identify dead plasma cells based on surface marker expression? I imagined the expression of certain surface markers would also change as cells undergo apoptosis ... can anyone tell me more about that?
Lastly, the obvious solution would be to pre-sort or enrich but I haven't gotten great enrichment with CD138+ kits (~20% PCs, working on optimizing) and sorting is expensive and rough on the cells so I was attempting to avoid it.
Thanks in advance for any advice/info you can provide!
Absolutely right, Dead cells take staining non-specifically so, if your cells are dear, you cant rely on them at all. You may not determine the plasma cell proportion from dead cells.
You may proliferate B sorted B cells by BAFF to increase numbers.
Absolutely right, Dead cells take staining non-specifically so, if your cells are dear, you cant rely on them at all. You may not determine the plasma cell proportion from dead cells.
You may proliferate B sorted B cells by BAFF to increase numbers.
You should not try to analyse subpopulations within dead cells, as autofluorescence, cell integrity disruption and aspecific antibody staining would make your findings unreliable.
If you standardize starting conditions (ie. absolute number of your cell of interest before treatment), you can evaluate the effect of your treatment at various time-points by calculating the relative and absolute abundance of your cell of interest on surviving cells (you should include counting beads in your FACS samples). You should also optimize culture conditions, as the inclusion of other cell types will most likely affect your results (both ways, as supporting or effector/competing cell can have different effects on your plasma cells).
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