Hello,
We have been running LDH assays on skin fibroblasts in conjunction with our MTT assays. Our hours of exposure range from 24h-96h. Media is aspirated and changed and cells are re-dosed with the toxicant of choice every 24h. Because the media is only collected for LDH at the end of the exposure period and because we changed media and re-dosed every 24h, we noticed a downward trend in LDH concentrations due to cell death at earlier time points. We worked with a statistician to correct this problem and it was suggested that we calculate our assays cumulatively. For example, 24h, 48h, 72h, and 96h results would be added together to take into account all LDH produced. However, the problem is that the LDH media is not collected throughout the assay but at the end of the MTT assay. Therefore, the results from the 24h exposure and the 96h exposure are from different assays. Hence, the % of cytotoxicity observed at each timepoint is not entirely linear due to intra assay variation. The question has been brought up if there is a better way to calculate. Has anyone here done LDH assays using differing time points while changing the media and re-dosing cells? If so, how did you preform your calculations?
Since you aspirate the culture medium (taking all LDH released within the period) at 24 h intervals, I do not see any better approach than add the the results from different experiments at 24h, 48h, 72h, and 96h as it was suggested.
Hi,
I hope that you'll find the notification on this old thread.
I'm planning a LDH assay and the timepoints are 0 ,2 ,6 ,24 ,48 hours. The protocol strongly recommends to perform the assay immediately after sample collection. My question is, is the maximum LDH activity sample to be collected in respect to each timepoint or at the last timepoint. That beats the instruction of doing it immediately.
Another question, the first step is to figure out the number of cells to be used in the assay by serial dilution of cells, does that also have to be performed for all timepoints?
I would keep the number of cells consistent for all time points.
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