My lab is experiencing unstable results with TF-1 cells and the CellTiter-Glo kit by Promega.
It's a very easy assay, seeding different amount of cells, growing for 3 days, then measuring via CellTiter-Glo. Strange thing is, the results are not constant, meaning we have triplicates and e.g. 2 wells are about the same value, one is about 1% of the other values. There is not even one seeding-concentration with all values about the same level.
On the other hand we tried MTT-assay. This showed very good and constant results! No extreme-low wells at all.
It it possible that TF-1 cells are acting strange with CellTiter-Glo? I don't think that it's a seeding-problem, a mulitchannel-pipette was used. And why is MTT giving such good results? Are TF-1 cells sensitive to shaking? We used NFS60 and Raji cells before, with fine results. We shake 15min then incubate for 15min, is this too much for TF-1 cells?
This is a luciferase dependent assays. Is the luciferase measurement equipment similar to the OD. In our lab we performed many luciferase measurements and sometimes, new technicians caused irregularities in different wells due to an other working protocol. For the measurement, cell lysis and adaptation towards light stabilisation is needed with the celltiter-glo procedure. So, we said shake the plates shortly, put it in the measurement equipment and let stabilize for 10 to 15 min before measurement, as this is a steady glow measurement. There is now need to measure within 30 seconds to 1 min. So, measure every min during 15 minutes and see what happens.This may solve the irregularities. If not, check if multiwell measurement is done and whether one of the photomultiplyers is out of focus or whether the well position is incorrect.. For instance fill lanes A, C, E, G with reagent and measure, than rotate the plate 180 degrees, and lanes A, C, E, G will be lanes H, F, D and B.
I hope, this solves your problem as the Celltiter Glo assay is very reproducible.
I agree with Willem. I find the key to consistent luciferase measurements is letting the plate stabilze for at least 10 min before measurement. As suggested, measuring every min for 15 minutes would show you if a particular well is not performing as well as the others or is the photomultiplyer tube is out of focus. Also make sure your instrument is set to read the proper plate configuration for the plate you are reading.
In my hands prolonging the incubation with celltiterglo to 1 hour at room temp resulted in better (lower) CVs and improoved parameters of my bioassay, using SP2/0 transfected cells
Thank you all for opinons and ideas. But the problem, I want to point out is that ONLY with TF-1 cells we get the strange results. With other cells like Raji/NFS60 the CellTiter Glo works fine, with same assay-settings...
Why we want to use CellTiter Glo is because it's more sensitive and the assay is much faster than MTT...
Any ideas?
Your reasoning for using CellTiter Glo are the very same reasons I use it instead of MTT. If it is only TF-1 cells, then I might ask if some component of the media is different from your other cell lines and if that might be interfering. Also, do the TF-1s lift off the plate if they become confluent? That might contribute to your variance.
it sounds like to me that your plate is off the position in the reader, I am assuming you were using a microplate reader. do you find the odd reading wells in particular position in the plate? may be the edge may be certain row or column etc.
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