I am analyzing CD8+ and CD4+ T cells in tumor tissue using flow cytometry but cannot detect IFN-γ. I did not use any stimulation. Reagents I used were cytofix, perw/wash buffer, cell staining buffer, and PBS. Should I use BFA as well?
Are you performing intracellular staining for IFN-g in CD4 and CD8 T cells? If yes, the detection is very low anyways. Try increasing the antibody concentration, use strong fluorophores like PE. CD4 may give you better expression of IFN-g as compared to CD8. Also, try detecting IFN-g in serum via ELISA to make sure your system is working. Good luck!
You should stimulate your cells and block the Golgi pathway (BFA/monensin/brefeldin).
Yes, you should be using BFA etc. Read the intracellular staining protocol appropriate to your antibody. I would not stimulate the cells as this obscures the baseline cell levels.
Mohammad Aqdas Yes fortunately, we did use PE as the fluorophore. Would probably do ELISA in the future.
James E Talmadge We did read the protocol, but they only mentioned BFA when stimulating the cells. But thank you for the advice I would definitely use the BFA
Will comeback to share the progress, thank you everyone!
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