We are planning to do LDH assay for different day(Day 0 to day10) experiment. The samples were already frozen at -20.C. We do have a Pierce LDH cytotoxicity assay kit. Its more or less an optimization. Can anyone suggest the protocol? Moreover, can we use the frozen sample? will thawing affects the LDH activity?
Hi,
I think that it is not possible. The principle of the LDH assay is that LDH is released by cells with damaged membrane (necrosis). Therefore LDH activity in the supernatant of the cell culture reflects necrosis. Thus, you need the supernatant to assess cytotoxicity.
I recommend you to use propidiumiodide or 7aad to test membrane integrity of your cells which also is a measure for detecting necrosis. However, also by freezing and thawing cell death is induced which might bias your results!
My recommendation is to always test cytotoxicity immideately, and not after freezing and thawing!
Good luck,
Robert
I was wondering the same, but with frozen supernatant samples. Do you think they can be used with this LDH assay?
Some time has passed, but I can add some experiences here. I tested the Pierce cytotoxicity LDH assay kit for frozen supernatants of BMMCs and LDH acitvity was detectable but was highly varying between the samples (0 - 70 % of complete lysis). I would not recommend to freeze more often than one cycle.
I also want to measure LDH activity from frozen supernatants. I would like whether LDH activity will lost?
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