Hi,
I am working with LDH-Glo (https://www.promega.com/-/media/files/resources/protocols/technical-manuals/500/ldh-glo-cytotoxicity-assay-technical-manual.pdf?la=en)
cytotoxicity assay and calculate % cytotoxicity from this formula:
% Cytotoxicity = 100 × (Experimental LDH Release – Medium Background) / (Maximum LDH Release Control – Medium Background)
I treat my cells (human pancreatic ductal cell line) with potentially cytotoxic compund which should damage them - and i get the excpected effect, however I do not understand why do I have such a high values in control group:
Triton X group - 100% cytotoxicity
Treated group - 60%
Non-treated negative control - 38%
I handle with the cells carefully and
Should I worry about so high cytotoxicity in non-treated group or just simply normalize experimental-treated values to seek only for drug-related effect?
Most appriciated if you could help me on this one!:)
Best regards,
Michal
Dear Michal,
I would agree with your idea to scale the cytotoxicity towards the values observed in the negative control. Ideally, there should be only little additional LDH activity in the medium when healthy cells are present, however it is possible that it is a characteristic of your cell line to secrete LDH activity into the medium.
If you suddenly got too high absolute activities in comparision with earlier experiments, try to check your cells for contaminations, which may compromize viability and increase LDH.
Also, make sure you set up your blanks properly; you must include serum into the blanks if you use it in the other treatments; it significantly contributes to total LDH activity measured.
Finally, if you suspect that there still may be a problem with the cells, consider double-checking your results with a second cytotoxicity assay; MTT and NRU are simple, cheap and frequently used alternative approaches.
Regards,
Patrick
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