I am using SH-SY5Y cells for studying cell viability by LDH assay (Kit from Thermoscientific). I seeded around 20,000 cells /200 uL media and followed manufacturer's protocol but did not get any red formazan colour. What could be the possible reason apart from low cell density?
Also, I would request to send me a step- by step protocol or precautions. Doubts like, should we add 20uL of sterile water (Spontaneous LDH) and 10X lysis buffer (Max. LDH) after removing the media or in the same media? Is it mandatory to centrifuge the plates? also needs to be clarified.
Thanks
Hi Sneha
The principle of this assay is measuring LDH level in the media. So, you have to keep the media and just add working solution and lysis buffer as in the protocol on each sample.
in the attached file, you will find the protocol i used before, no need to optimize the cell concentration.
Good luck
I would do the max LDH sample, it can essentially serve as a positive control for color development. Also spin it will collect the dead cell debris which can interfere with the assay.
What kind of injury did your perform, could it be that your cells did not reach the threshold for injury to release LDH?
Always follow the protocol, especially when you are not sure of what you are doing.
hello Fawaz,
You said we need to add the lysis buffer to all the samples. So if we do that then definitely the LDH will be released by all the cells, which upon addition of lysis buffer will be dead (even the healthy ones), then how do we differentiate the cell viability among samples????
Don't add lysis buffer to all wells, then all your samples will be lysed. Just seed 3 wells with cells, then use those as your maximal lysis.
Basically, for all triplicates that are not lysed (measured for sponteanous LDH release), you have a max LDH release well that corresponds to it.
See figure 2:
https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0011851_Pierce_LDH_Cytotoxicity_Asy_UG.pdf
Alternatively, you can use one of your samples as your baseline, which would mean that you don't have to have maximal release wells. This can be your control group for example, which would be set to = 100% and your "treatment" groups can be expressed to relative to the control group. For example if the LDH release was 0.50 in control and 1.0 in the treatment group, than you have a 1-fold increase in LDH release (vs. control). This can be arguably more practical and applicable especially in scenarios of acute or mild injury.
Thanks Keissling... I got the results today. Yeah I didn’t add lysis buffer to all except three wells taken as triplicates.
Funnily in the last experiment I had forgotten to mix the assay buffer which failed the experiment, making me think otherwise until I figured this out.
Thanks for your suggestions though, they were helpful.
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