I am trying to perform a modified MLR adding CD14+ monocytes to CD4+ T cells (both positive MACS isolation), under different conditions. However at 5 days, even with HEPES in culture media, the wells are yellow and I'm seeing significant CD4+ cell death.
I have been spiking the media with varying volumes of fresh media at intervals throughout 5 days however this doesn't change the outcome much.
Should I spike the wells with new media?
Should I completely exchange the media?
Could this be due to the ratio of APC to responder cells which appears to be crucial (currently 1:1 or 1:2 CD4:CD14)
Any other ideas?
Lastly I am also labelling the CD4 cells with CFSE to check for proliferation, is it ok to do this prior to the 5 days incubation or should this be performed at end of 5 days?
Many thanks
Hatty
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