Hi, I am wondering whether anyone else has problems with this kit from Promega?
The issue is a huge variation between replicates in this assay, the standard deviation and %CV being unacceptable every time. Our whole team tried to use or optimize throughout 3 years and the results are still unusable. So many boxes of the reagent wasted.
To be clear, the issue happens despite changing: the operator, the lab, the plate reader, reagents batches, cell line/PBMC source, cell density, incubation time, using cell strainers... It is also not a well effect or well (luminescent) cross-talk and we do not use the wells on the plate perimeter for the tested samples.
I tried to ask Promega for troubleshooting advice but they ignored the emails. Obviously we neither rely on nor recommend this kit anymore, but I'd like to solve this mystery. Do you also have this issue or can suggest any ways to improve the result consistency?
Thanks for your time!
Without being an advocate of Promega we never had problems with the Cell Titer Glow. Two things to recommend: i) have you ever tried any standard and robust cells others than PMBC to qualify the kit (using cell strainers can cause a huge variability !) and ii) have you tested any other viability kit like Resazurin or MTT? This might help to trouble shoot.
If you are working with cells a lot you should check-out Cellseeker https://www.cellseeker.org/). This is an easy and free to use app to organize and inventory your cell collection online.
I know this was asked almost a year ago, but in case anyone else runs into this same problem:
Some cell lines are difficult to plate evenly (clumpy, super small and hard to count, etc.), or in my case, different lines have uneven viability post plating. There can also be settling of cells in the tube/reservoir from which your cells are being pipetted, which can lead to variation in plating density between the start of the plate and the end of the plate. I differentiate neurons in situ in 96-well plates from stem cells, which takes several weeks. Over that time, the number of cells that die off in any given well varies quite a bit. Since the ATP assay requires the cells to be lysed, it's not really possible to do any other assays with that same plate. So I make a clone plate and measure total protein content in each well of the clone plate, then normalize each ATP plate well to its corresponding well in the total protein plate. If you want a really quick viability test, you're better off with another method, but this works really well for me when I need a really accurate measurement.
To make the clone plate, I have a repeater 200 uL multichannel pipette that can plate 100 uL of cells x 2 per aspiration. I set two 96-well plates side-by-side in the hood and plate the first 100 uL of cells into the first plate and the second into the same row on the second plate. For the first plate, I use an opaque white luminometer plate, and for the clone plate I just use a standard, clear 96-well plate. The assay I use for protein is the SRB assay, which has worked well for me, but you could just as easily use a BCA assay or whatever. I suspect you may just need something to normalize your data to. This method has significantly cleaned up my results.
I plate primary NK cells in 96 well opawue plate, and treat with monoclonal antibody (at 10 different concentrations; 3 known to kill, 3 doesn't kill, to generate dose-response) to cause apoptosis. However, the same method/lot of cell titer glo 2.0 reagent, same everything yields strange differences. Sometimes the dose-response is perfect, and sometimes the untreated wells and treated wells show absolutely no difference in luminescence readings. At first I thought it could be that the antibody went bad, or antibody preparation was bad, tried all ways, but it is still mysteriously random why it works sometimes and why it doesnt sometimes! Would love this mystery solved! And yes, promega ignored me too!
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