I'm trying to feed IFNg stimulated RAW macrophages (100ng/mL) with a range of OVA concentrations (incubated overnight, whole protein from Sigma) and then adding the DO11.10 cell line (not from mice) to these cells (I've tried fixed and unfixed macrophages). After 24/48h I was unable to detect any IL-2 secretion by ELISA. I confirmed the DO11.10 cells were able to secrete IL-2 by coating a well overnight with CD3/CD28 and they produced very well at both time points. Would anybody have other suggestions? Should I just try staining the cells for activation markers instead of looking at IL-2?
HI,
There is a thread below you might want to check out. It seems this individual had a similar question.
https://www.researchgate.net/post/Does_anyone_know_a_good_protocol_to_generate_OVA_peptide-pulsed_dendritic_cells_DC_in_vitro
You can start by adding a control where Raw cells would be pulsed with the OVA peptide recognized by DO11.10. If it works well, then the absence of detection when the full length OVA is used is likely due to limiting OVA uptake/processing. If it doesn't work, then either MHC II expression on Raw cells is a limiting factor or your hybdridoma is not sensitive enough. You could also use B cells from Balb/c mice instead of Raw cells as a control.
Thank you very much. I did try the uptake of full length OVA over a range of concentrations with no luck. I'll probably try our one of our B cell lines which can interact with DO11.10 cells, which others in the lab have had success with previously. I just hoped IFNg treating the macrophages would upregulate MHCII enough that it would work.
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