the LDH level started to increase with increased drug concentrations, but the higher concentrations gave lower LDH levels compared to the smaller ones. Is it because the higher conc. resulted in cell death in the majority of cells? or there are other reasons? putting into account that, treatment duration is SAME for all of them
It is better to reserve 2-3 wells to set 100% cell death with tritonx100 (1% or 2% final concentration ) or SDS and check LDH in your system.
It is a matter of cell growth. I presume that you are treating for 24 hours or longer in a dividing cell line. At toxic concentrations, the total cell number would decrease due to cytotoxic or cytostatic events. At the lower concentration, you have enzyme leakage from a damaged cell population that is increasing in cell number due to cell division. At high concentration, you have enzymes from cells that are killed after initial treatment with little or no cell division occurring.
I think that what you obtained is quite unusual but, from my experience, LDH can be not the best way to assess cytotoxicity for prolonged exposure beacuse LDH in medium have an hal-life of about 9 hours so when you perfomed the LDH assays you will measure the enzme released in the last 9 hours. In different experiments we so that the total amount of LDH detected after 72h or treatment were lower than levels detected at 24 hours while data from MTS assay (cell proliferation) confirm that there were less live cells in 72h treated wells respect to the numner in 24h cells.
For citotoxicity, try other methods such as adenilate kynase by Lonza or DNA release by promega (cell tox green).
other suggestion is to couple citotoxicity with proliferation assays.
Thanks all.
But still, my point is, I know that the LDH level will be decreased with time, but this is not the case that the question target, so my point is, I got positive results from all the concentrations that I used, those results indicated increment of LDH level that proportional to increased concentration, till reaching specified one, after that the level of LDH strats to decline, but still more than the negative control, but less than the other concentrations. What is the explanation for this
Thanks again
Dear Nikola, thanks for passing through. Yes Albert gave a logical conclusion, and I'm also satisfied with it, so at the ennd we can say, it is a matter of cellular number after the treatment period.
Thanks again
It depends on the experimental system you are using. Are you using cell lines and if yes, which cell lines?
It also depends on type of drug you are using. Which drug are you using?
Are you measuring activity of LDH that is released out by the cells and/or activity of LDH in the cells/organ (i.e. extracted from cells/tissue/organ)?
LDH activity is often linked with maintenace of NADH to NAD+ ratio and substrate level phosphorylation.
Increase/decrease needs to be viewed by taking into account several factors together.
@ P. Pardha-Saradhi
could you please give specific factors that could cause this phenomena?? taking into account that I'm using normal liver cells, for measuring the LDH that released from the cells as a hepatotoxicity parameter
Thanks
Dear Hozeifa Mohamed Hassan
I consider that this related to cell growth and if you try dividing cell lines for over 24 hours to toxic concentrations, there is a decrease of cells by cytotoxic events. When the concentration is lower, has lost of enzyme in damaged cells and no increase in cell number.
I agree with Diego. I think it is not the best method to measure citotoxicity. You need ti combine another method to measure proliferation.
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