In order to measure cytotoxic activity of T cells, we are trying to set up the DELFIA Cell Cytotoxicity assay. After many different attemps, we are still dealing with a high degree of spontaneous release of the TDA ligand.
In our latest protocol we do the following. We stain MC57G mouse fibroblasts for 15 min at 37C with 1.5 uL BADTA in 1 mL culture media. After we wash the cells 5 times with culture media with 1 mM sulfinpyrazone or 2.5 mM probenecid added. In the second to last wash, we incubate the cells for 30 min at 37C in the wash buffer to allow some TDA to be released and washed away. After the 5th wash, we seed 10K cells per well. We use 1% Igepal 630 for maximal release.
The results of spontaneous release based on the maximal release:
Culture media only: 63%
1 mM sulfinpyrazone: 46%
2.5 mM probenecid: 61%
In our eyes these values are all too high. sulfinpyrazone showed the lowest spontaneous release, but is still 46%. We are wondering what changes we can make to the protocol to reduce the spontaneous release by the cells. Or would this be a problem that is specific to the MC57G fibroblasts? Please help, thanks in advance!
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