I did LDH cytotoxicity assay on MKN-1 cell line with my compounds at various concentrations. After 24 hours of incubation, LDH assay was performed according to manufacturer's protocol. There is something strange that my un-treated cells have more LDH cytotoxicity (%) than some of the treated samples (not all). How it could happen? If happens then what is the reason?
My cell condition is very good and they grow in monolayer and there are always some apoptotic bodies when they become over-confluent. Is this due to that phenomenon? But as far as I know LDH positive result is due to membrane rupture (probably due to necrosis but not apoptosis). Please enlighten me. Thank you.
Thanks Dr. Tanya! Can you please recommend some assay without such pitfalls?
Thank You Dr. Tanya! I have one more question. If cells detach after applying compound then which assay would be suitable (as LDH failed in my case for some molecules)? Crystal violet needs attached cells.
Hi,
are you sure that you did the calculative evaluation correctly? Remember that in LDH, the signal-viability relation is inverse to what is observed in most other cytotoxicity assays (MTT, NRU, ...), thus high signals mean more toxicity resp. less viability in contrast to other assays. Likewise, be sure to calibrate 100 % toxicity (not viability!) via the positive control.
Apart from that, generally if cells have detached from the vessel, the membrane should be compromised and the usually intracellular LDH should be in the supernatant, so this should not interfere with your assay.
Dear Patrick! I exactly followed the procedure i.e. more signal in LDH means more toxicity unlike MTT etc. I also calibrated the 100% toxicity using Triton.
My cells adhere very strongly and only detach in the presence of chemicals. So I am wandering that why control cells have more LDH signal as compared to some treated one. The chemicals in treated ones have no cytotoxic effect so it must be either higher than control or atleast equal to the control but not less than control. So, I am wandering why? Do you have some explanation for that one?
Hi,
ok, you said that your cells detach from the vessel, but you do not observe cytotoxicity in any treatment? This is somewhat contradictory, as usually, if cells detach, they can be considered either dead or in a late stage of dying (e.g. via apoptosis). However, there are exemptions for compounds specifically disrupting the cytoskeleton, where cells may detach but still remain perfectly viable. In this case, you may run a different test (e.g. MTT) using a protocol adapted for suspension cells, which essentially means you collect all (including the non-adherent) cells in the medium by centrifugation first.
Apart from that, enzyme inhibition or denaturation by your compound (e.g. heavy metals, detergents, solvents, oxidants, ...) as already suggested may explain your issues.
Also, I assume that you made sure that all wells including blanks received identical carrier solvent and FBS concentrations? LDH activity originating from FBS supplementation can also cause problems if not properly corrected for.
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