Have anyone used the WST-1 assay for ID8 cells?
Is 1000 cells a good starting amount to seed them?
Can I induce cell death to my cells using Triton X and then use the WST-1 assay after to measure viability? Because I read that Triton X is used as a stopping solution, so I don't know if it will interfere with the reaction.
Hi Bruna,
We use ID8 cells pretty regularly in our lab. They grow pretty fast. I haven't specifically done a WST-1 assay on them, but we generally seed them at 500 or 750 cells per well on a 96-well plate and they are 90-100% confluent in 4 days. Granted, we are transfecting them for a non-colorimetric read out, so keep that in mind.
Also, if Triton-X interfering with the assay is a concern, you could always use another colorimetric death assay, like a MTS or MTT assay and compare it with your WST-1 results. I realize that they assays aren't cheap, so maybe you can ask around your department to see if someone in a neighboring lab can lend you some.
Anyway, I hope this helps. Good luck in your research!
All the best,
Bryan
Bryan, thank you for you answer! Really helpful!
So I am planning to do both WST-1 and MTT methods, to compare which one is better. But I am afraid I will have to standardize the amount of cells used, right? So I've read in the literature that for the WST-1, 1000 cells are enough to start. However, the manufacturer recommends that for the MTT assay, I seed at least 5000 cells. Do you think it will be a problem if a use 1000 for wst-1 and 5000 for mtt? - In terms of comparation between them.
Hi Bruna,
You could attack this two ways.
1. You could do a preliminary small scale cell titration. You could set up 500, 1000, 2500, and 5000 cells/well in triplicate. Then, treat your cells with the compound of interest and run both assays. If you keep it small enough, you could run both assays on the same plate. This is just to give you an idea of optimal conditions and how fast your cells grow. Then, you can scale up and run 2 full 96-well plates in parallel for each assay with the conditions that work best (which may or may not be standardized).
2. Honestly, 5000 cells/well for a 96-well plate for ID8 cells sounds WAY too high. They would overgrow the plate too quickly. The other thing you could do is just use 1000 cells/well for each assay. I think it would still work, but even if it doesn't you could just redo it with a higher cell density.
Anyway, it's up to you to decide which approach is best for your experiment. I think generally, the only difference would be how fast the assays develop in proportion to starting seeding density. For endpoint assays like this, it might not make too much difference, as long as your final readout is comparable between assays.
Again, best of luck in your research!
-Bryan
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