I have encapsulated my cells using chitosan at different concentrations of 1mg/ml of 0.1M acetic acid, 1mg/ml,0.1 mg/ml. I run MTT at different timepoints of Day0,Day1, Day3 and Day7. at Day 3 time point the formazan formed didn't seem to dissolve in isopropanol. what might be the reason behind this. I could see the proliferation rate was high. Im using a cell line that grows really fast
Hi Akhela,
My lab uses MTT extensively to quantify cellular proliferation. We use the following protocol :1) Remove cell media with an aspirator, and incubate the cells with MTT stock solution for 2 hours (MTT stock solution: 5 mg/ml dissolved in Fluorobrite Media filtered though 0.22 micron filter) . We typically use 75 microliter of MTT solution per well in a 48 well plate. 2) Following this incubation time, we add the Acidified IPA Lysis buffer (0.1N HCL in IPA) to each well (1:5 ratio -> 75 microliter of MTT and then add 300 microliter of lysis solution) and place the plate on a shaker table for at least 30 minutes. If the cells are very healthy and create a lot of formazan, then this time point is increased until all the formazan is dissolved. For extremely healthy cells, this process of formazan dissolution could take 1.5 hrs or so. If it does take this long, make sure to plastic wrap the plates to reduce any errors that occur as a result of IPA evaporation.
Hope this helps!
-Steve
Hi Akhela,
when and how do you acidify the isopropanol? From your protocol it seems that you are incubating the cells in presence of chitosan and acetic acid, and then, once incubation is over, you add plain isopropanol to the cells.
My advice is to remove the wash the cells after incubation, add MTT, gently remove the medium and eventually add isopropanol acidified with HCl (1:1000-1:500 v/v). That should do.
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