Hello,
We are doing LDH assay (roche) using PC12 cell line (20 thousand cells in each well) with NGF. We are testing a compound with different concentration but in each concentration our result becomes negative because of higher negative control than treated ones. We are using this formula,
Relative LDH release = ( X - Negative control) / (Positive control - Negative control)
Where X = sample absorbance value (@ ~ 490 nm)
In negative control, there is only cells with low serum medium nothing else.
What would be the reason for this reduction in treatment groups?
Thanks in advance.
Some chemicals interfere with LDH assay development. Take a Triton X100 control and sample it with and without your test drug to confirm that it isn't inhibiting color development on the biochemistry of the assay itself.
I think that Jordan suggested to compare two groups of cells, which I also recomend to do:
1. Tritton
2. Tritton + your drug
If group number two gives lower absorbance than group number 1 it means that your drug causes chemical interaction with the assay itself, thus giving false negatives.
Thank you Jakub for clearing that. I didnt think about it. It makes sense and we will try thank you for both of you. I will let you know about it.
Thanks again
Hi Suleyman, did you manage to fix your problem? If so, please do share what you have discovered, as I am also facing the same problem at the moment. Thank you
Hello.
Did you manage to find the solution to your problem? Because I am facing similar issues with my LDH Assay!
Thanks
Hi Faria
I used this suggestion made by Jakub and it worked:
1. Tritton
2. Tritton + your drug
If group number two gives lower absorbance than group number 1 it means that your drug causes chemical interaction with the assay itself, thus giving false negatives.
Hello Thanks a lot for the suggestion. I tried that as well but with the positive control provided to me in the LDH Thermoscientific kit instead of Triton X and the value reduced with my compound. It means for my compound I can not use LDH assay to monitor cell death? Or you know some modified protocol to make it work?
Thanks in Advance!
Dear all, I am facing the same problem. Can you please share your experience? Is there any better assay to check the cytotoxicity in case of incompatible of certain compounds with LDH dyes?
Faria Khan what alternative assay you did instead of LDH as my compound also interfere with the LDH assay.
@numan
I have switched to MTT assay for viability and to check cell death you can go for etbr or PI staining with calcein AM counter stain for viability. You can use fluorescent micfoscope or fluorometery or facs for analysis. You can get similar results with almar blue dyes or xtt assay to check viability. Apoptosis can be measured through facs so if u have that available, best option is to switch to that even though it's not high through put assay but it can give you quantitative measure of necrosis/ apoptosis compared with control.
Faria Khan i have already done the MTT assay but i thought it would be great if i confirm it through LDH as well. I have annexin kit available right now. Do you think if LDH has issues so does annexin will have ?
@numan I haven't worked with Annexin V so can't be really sure if it will work for you or not.
You can also try to see the pH of your compound with PBS or media first. If your compound significantly alters the pH of your media then it means there will be a lot of interference if you try to run any assay in the media/ PBS. In MTT we stain the viable cells while in LDH assay we use the media to determine percentage release of LDH in the media so factors in media are important. What's your protocol for Annexin V? Annexin V is commonly used for distinguishing early apoptotic cell from apoptotic/ neurotic cells in Flow cytometry analysis when counter stained with propidium iodide. Annexin V will also stain those cells that has some early signs of cell membrane integrity loss. So in your case, just try to run a trial assay and see if you would be able to get results. Otherwise propidium iodide staining can give you idea about cell death as well because PI only stains DNA (And a little of cellular RNA) once cells undergo apoptosis
@Faria khan,thanks for the suggestion. I will try to for staining if I still having issues
I have the same problem, the negative control reading is higher than my killer group. It may be caused by high cell density and need to pepeat determination of optimum cell number for assay. I will try lower cell number in experiments.
By the way, dose any friend know what is the optimum N2a cell # for LDH assay?
- Badges
- Science method