Live cell imaging using IncucyteZoom is the best way to measure effect of drugs on rate of cell proliferation. I noticed that despite of how accurate we try to count the cells using automated cell counters (countess) sometimes not all the datapoint do not start at the same point.
If we normalise the data to timepjoint 0 the gradient of the slopes changes which is not ideal.
One researcher stated I should seed cells at different densities and introduce an error in counting and choose the densities that start at the same point which I believe is not correct because you are just increasing additional errors to that of countess and additionally assuming all cells have the same size and shape.
I had been deducting the baseline values of time-point 0 from all time points of respective condition such that all points start at 0. Reason: This deduction will not alter any slopes.
The only problem is when cells reach 100% confluence and plateau deduction of baseline for example: if the confluence started at 20% at 0 time point and reached 100% at 96 h and remains plateau unto day 7 , deducting the value of baseline will show that the cells reached plateau at 80% at 96 h and 120 h. If I plot the data only in the log phase and deduct the baseline I think is the best option.
Can anyone please comment on this and help me out.
Using cell counting to measure proliferation rates is inherently limited by the affect of cell-cell contact inhibition. You should be able to overcome most of the error that slightly different starting confluencies may cause by using a larger number of replicates. However, I would advise you to use a BrdU or EdU assay instead, which measure the proportion of cells that proliferate within a shorter window of time. The built-in analysis modules of the Incucyte should be able to perform this assay easily.
This has been bugging me too for some time now.
There are some examples of cell proliferation incucyte data in the literature whereby data for each condition are normalised to its respective starting confluency at the first timepoint. I've previously consulted an experienced researcher who uses incucyte a lot for their work and they have published data using this method, so it seems a perfectly reasonable option.
As long as you're comparing apples with apples, it should be fine.
David J Walker is it possible to cite the publication in which you found informations about normalisation please
Thanks
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