I'm currently working with a BT474 cell line, but my cell line was confluent, and I forgot to wash it through PBS, thus I trypsinized it. As a result, even though I trypsinized my cells, they remain stuck to the culture flask's surface.
Give your thoughts on the mechanisms behind PBS/DPBS washing before a bio-assay or removing adherent cells from a culture flask.
Thanks in advance.
Hello Md. Shahidul Islam
Trypsin is inactivated in the presence of serum. Therefore, it is essential to remove all traces of serum from the cell monolayer, and this is done by washing the monolayer of cells with PBS (without Ca2+/Mg2+). When you failed to wash the cell monolayer with PBS/DPBS, the action of trypsin was inhibited by serum traces present in the cell monolayer. So, the cells remain adherent to the surface of the flask because the action of trypsin was incomplete.
Best.
Hello Md. Shahidul Islam
Trypsin/EDTA cannot function well in cutting the adhesion molecules (cadherins) in adherent cells in presence of serum (FBS) in DMEM/EMEM. That's why PBS washing to remove FBS prior to trypsin addition works well in cell separation.
Dear Islam
FBS contains protease inhibitors particularly α1-antitrypsin, which inhibit the trypsin activity. Therefore you have remove the FBS before trypsinization
Best wishes to Malcolm Nobre, Salequl Islam, Manoj Wijesekara all of you for your response.
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