Trypan Blue and Hoechst staining.. - am I doing it right?? please help.... (Apr/02/2009 )
I want to study cell viability and detect apoptotic cells in a cancer cell line when exposed to hypoxia. So I use varying concentrations of cobalt chloride at different time points. So this is my plan:
For Trypan Blue:
seed 1 million cells/well in a 6 well plate 24hrs before treatment. The next day add cobalt chloride and then extract cells at diff time points and do a trypan blue staining to find the ratio between dead and live cells.
Question: I know that trypsinising the cells and resuspend in pbs and trypan blue and counting in haemocytometer would do the job. But I thought maybe, wash the cells with PBS and then add the PBS+trypan blue into each well and incubate for 3-5 mins and wash off trypan blue and resuspend in PBS and look under the microscope. Say I count 200 cells per well, and find the ratio of live to dead cells. This way I can take a picture of my cell staining and have it in my thesis or even count them how many ever times I need. Am I making sense? Also if I just cound 200 cells and if I want to do a similar experiment but using lots of cells, can I take into account this ratio (from 200 cells) and scale it up and expect similar results??
For Hoescht staining:
seed n number of cells (not the same number of cells in every well) into 6 well plates having cover slip and let it grow for 2 days. 3rd day, get rid of media and fix the cells usinf formaldehyde or clarkes solution and lift the cover slip off and transfer to new plates(the small ones) or leave them in the same dish and treat with hoechst stain for 30 mins and then add mountant solution onto a glass plate and place my coverslip upside down (over the moutant) and let it dry and then look into the microscope for some pretty pictures.
Question: Will the stain remain for n number of days without affecting the live cells? that is, can I store this glass slide for long and look at the nucleus if in doubt?? Can I not use the cover slip and do the staining in the well plate itself and just capture the image from the microscope and count 200 cells using imageJ software and find the apoptotic index using = 100xapoptotic cells/200 cells. ???
Plllllllleeeeeeeeeeeeeeaaaaaaaaaaassssssssssseeeeeeeeeeeeeeeeeeeeeeeeeee advice... Theres no one in my lab who can help me with this regard!!!!!
I saw your question, I think it would be better if you used the annexin or the propidium iodide which are more precise for the detection of cell mortality.