I am trying to isolate B-cells from the spleen of a STAT3 KO mouse and plan to stain with Annexin V and 7-AAD. I will then use flow to determine apoptosis and overall cell death. I have used Staurosporine in the past; however there was no distinct difference between the cells that were treated with Staurosporine, LPS, and non-treated. I am trying to figure out if it is the problem is the timeline, the treatment, or the staurosporine. Would love some help!
Thank you!
Dear Luciarita!
Please You look at this article:
Article c-Rel gain in B cells drives germinal center reactions and a...
Flow cytometry and imaging flow cytometry
For staining of apoptotic cells, the CaspGlow active caspase staining kit (fluorescein, BioVision) was combined with 7-AAD and the Annexin V apoptosis detection kit (APC, eBioscience).
Dear luciarita
Check both the staurosporine concentration and the treatment timeline, staurosporine should work as a positive control.
If you need another strong positive control, you can use H2O2 or other drugs such as etoposide or doxorrubicin. An overnight treatment with 20 uM etoposide should be enough.
In case of Staurosporine, maybe the concentration you employed is higher. Usually, doses lower than IC50 will be better for special incubation time.Also, for apoptosis, you can use mitomycin c and 1% H2O2.
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