Protocal for DAPI/ Hoechst staining adhering cells - (Nov/30/2005 )

Hi, my HEK293 cells seem to have problem, I would like to do a quick staining with DAPI or Hoechst to exclude the possiblity of mycoplasa contamination.

Can I just stain the whole dish cells instead of transfer the cells to the coverslip? Could you please tell me how to fix the cells and stain the adhering cells with DAPI OR Hoechst? Thanks!

Snow-protein

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Hello, what I usually do when testing my cells for mycoplasma contamination is staining them directly into the flask or plate, incubating them with DAPI for 5 minutes before I take the plate to the microscope (off course I wash and fix my cells before). I don't transfer the cells to a coverslip but it depends of your microscope because to see through a deep surface you will need an inverted microscope.

Hope you don't have mycoplasma, good luck

Pipo

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I agree with pipo.
i wash twice my plated cells with PBS, then I fix them with PFA on ice for 15 min approximately then I wash again twice and then I add my Hoechst for another 5-10 min. I check them under the microscope and as soon as I see blue cells I wash away my Hoechst and I keep my cells in PBS
Good luck
Maria

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Thank Pipo and Maria very much! Both your suggestion do help me a lot!


Snow-protein

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