The aim of my experiment is to see if a kinase inhibitor reduces cancer cell viability. I am using 2 different cell densities 50,000 cells/well and 100,000 cells/well and different doses of the kinase inhibitor.

  • $\begingroup$ What sort of research or thinking have you done? What could be some possible reasons for different plating densities? $\endgroup$ – MattDMo Jan 5 '17 at 23:05
  • $\begingroup$ I have been told to plate some of the wells at 50,000 cells/well and others at 100,000 cells/well, but haven't been told why. In all the papers about colormetric assays I have looked at only one plating density is used. $\endgroup$ – Bio124 Jan 5 '17 at 23:30
  • $\begingroup$ I have read that the lower the cell density per well, the more greater the effect of drug treatment. $\endgroup$ – Bio124 Jan 5 '17 at 23:51
  • $\begingroup$ Also at higher cell densities the metabolic state shifts from proliferative metabolism to quiescent metabolism. $\endgroup$ – Bio124 Jan 6 '17 at 0:07

The comments you have given are some reasons, although they also depend on the confluency of the cells. Your teacher/professor/advisor is also making sure that you can see an effect at low levels of drug, as well as making sure that changes in the signal are visible - for example, does a 50% reduction in viability due to drug activity give the same drop in signal as reducing the number of cells by 50%? You don't say what kind of assay you're running, but a fairly common and inexpensive one is the MTT assay, with some kits nowadays switching to XTT for greater sensitivity and a reduction in the number of steps in the assay (example kit from Cell Signaling compared to an MTT assay kit from ThermoFisher), as the final colorimetric product does not require solubilization before reading. Finally, depending on the lab you're in, the conditions for the assay may not have been optimized yet, so your instructor wants you to test a couple of different densities to see what works best.

  • $\begingroup$ Firstly thank you for answer, I really appreaciate it. I am still a little confused though, are you saying that if there is a 50% decrease in viabilitiy in 50,000 cells/well experiments at a given drug dose, then there should also be a 50% decrease in viability in 100,000 cells/well experiment at the same drug dose? In both my experiments (50,000 cells/well and 100,000 cells/well) I haven't reached a 50% reduction in cell viability. $\endgroup$ – Bio124 Jan 6 '17 at 16:36
  • $\begingroup$ @Bio124 no, what I meant was if there was a 50% reduction in viability of the 100k wells, the signal should be the same (or very similar) as the control 50k wells. It was just an example, don't worry if you're not treating with a high enough concentration of drug to hit 50%. If you click on the Cell Signaling link and look at the first data figure, you'll see how the viability curves vary at different plating densities, although in this case they're looking at signal over a period of time (as the cells grow and multiply) instead of varying the drug concentration. $\endgroup$ – MattDMo Jan 6 '17 at 18:56

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