I am trying to perform colonogenic assay on MDA-MB-231 cells. After 14 days of treatment, I wash cells with PBS and add 0.1% crystal violet in methanol to the cells. I stain them for 30 minutes in dark, aspirate the staining solution and allow them to dry for a while. I can see colonies under the microscope but the picture of crystal violet stained wells don't look that good as they look in the literature (https://www.google.com/imgres?imgurl=http://www.nvrb.org/wp-content/uploads/2016/10/nprot.2006.339-F1-730x410.jpg&imgrefurl=http://www.nvrb.org/radiation/&h=410&w=730&tbnid=7Hs-ObOXWJc4QM:&tbnh=160&tbnw=285&usg=__CgPe7hrvOYNM40m5yH0l39Xms5s=&vet=10ahUKEwjWj8acg93TAhXM34MKHTSqDvQQ9QEILjAA..i&docid=ozQCLykr-IJxsM&sa=X&ved=0ahUKEwjWj8acg93TAhXM34MKHTSqDvQQ9QEILjAA#h=410&imgdii=7Hs-ObOXWJc4QM:&tbnh=160&tbnw=285&vet=10ahUKEwjWj8acg93TAhXM34MKHTSqDvQQ9QEILjAA..i&w=730).
Where am I doing wrong?
Your picture doesn't look especially bad, I can see colonies just looking at it. That being said: crystal violet protocols can require some optimization. Try coating the plate for different lengths of time - I rarely use more than 5 minutes, myself. It can also help to wash it off more rigorously, which you don't mention doing at all - just wash in PBS, or even DI water, a few times, maybe try letting the final wash sit on the cells for an hour or so. And turn the flash on when you take a new picture, so the image is brighter.
Finally, do you have to use 231 cells? They don't form the nicest colonies in general - another cell line might give you prettier pictures.
Thanks William and Zemin, Yes, I didn't wash it after crystal violet staining. I was afraid if the washing would wash away the crystal violet stains. Thanks for the suggestions.
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