Murine splenocyte stimulation trouble
I’ve been doing experiments where I culture mouse splenocyes or human PBMCs in anti-CD3 coated plates (for 18-24 hours, so pretty short), then stimulate with various compounds short term (5 hr) to assess interferon gamma gene expression changes via qPCR and protein via flow cytometry. From the flow data, IFNg production is increased in CD8+ T cells from both human and mouse cells with a specific compound. A few months ago, I was doing gene expression studies using the same protocol and am now having trouble seeing consistent IFNg expression between mouse experiments specifically. The human PBMC cultures are very consistent with IFNg gene expression after 5 hr compound stimulation. But on one day, with one mouse spleen, the results would show increased IFNg expression, and the next day different mouse, I would see no changes at all. RNA quality and housekeeping genes looked golden, just a total absence of any effect. I tried so many things like switching FBS lots, different cell density plating, sampling at earlier time points (the compound stimulation is 5 hours), etc. I ended up isolating pure mouse CD8 + T cells from the spleens and doing the same experiment a couple times, and found that IFNg gene expression was more consistent and peaked at an hour post compound addition. But I have gotten extremely convincing IFNg expression at 5 hours from the mixed splenocyes and cannot for the life of me figure out WHY it‘s so tricky to get consistent results each time. Does anyone with experience with mouse splenocytes and mouse CD8+ T cells gene expression studies (particularly for RNAseq) have any suggestions for how to improve consistency between experiments? Or is there anything else that I am not considering which may be at play here?