I want to avoid buying expensive LDH cytotoxicity kits (~1,- €/well) and thought that probably other people already had the same idea before. I want to use the assay to assess cytotoxic effects of natural compounds on tumor cells in a 96well format.
Can anyone provide a protocol, or helpful comments on this?
Hi kai, I am sorry, I have always trusted the 'expensive' kits. I did look for an alternative a few years ago but could not find anything reliable. Maybe through this forum, we can get some solutions. All the best.
Worth discussing with Vicki Stone's group at Heriot-Watt. I know they developed their own LDH protocol for assessing nanoparticle toxicity. Best wishes.
Hi all,
who ever is interested in a self-made LDH-Assay protocol, don't hesitate to contact me.
Best Kai
P.S.: @Margaret Saunders: Thanks for the advice to contact Vicky Stone. I got the protocol within a day! Thumbs up
Dear all,
I am trying to establish a self-made LDH protocol in our lab according to Article An optimized lactate dehydrogenase release assay for screeni...
The LDH assay itself seems to work very well.However, the lysis of adherent cells for the maximum LDH release does not work reproducibly. When I add 20 µl of 10% Triton X solution in H2O to the cells in 200µl of medium in a 96 well plate, mix 15 at 500 rpm, incubate for 45 min at 37°C, mix again and take 25 µl for the measurement, the triplicate values vary a lot. Any idea what is going wrong here. Is it really necessary to pipette the cells up and down? The original PIERCE protocol works very well without any additional mechanical disruption.
Another issue is the stop solution, which is 1 M acetic acid according to the protocol. When I add it to wells containing medium but no cells the color changes immediately which indicates probably means that the final pH is too low. Should I reduce the volume of the acid or rather go for another inhibitor of LDH activity? Copper sulfate is an inhibitor of LDH, however here I am not sure about the final concentration. My current guess is 1 mM. 50% dimethylformamide and 20% SDS at pH 4.7 diluted 1:1 with the reaction mix should also block the enzymatic activity. Since we do not have Dimethylformamide in the lab, I would use SDS only dissolved in water. Do you think the pH of the stop solution is critical at this step?
Many thanks for your help/suggestions!
Valeria
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