Determining the % of GFP/CHERRY cells in co-mixed culture - (Jul/18/2012 )
I am co-culturing cells labelled with GFP and cells labelled with CHERRY. I want to determine the percentage of each colour before and after treatment with a compound. FACS is not an option. Does anyone know how this can be done?
Any advice would greatly be appreciated.
You can simply seed the cells onto slides or coverslips, or even in the flask if you have the right sort of microscope, and do a count of total cell number, the number of GFP and cherry positive cells, then compare. It is probably best if you can take pictures and count the cells from that rather than trying to count directly under the microscope.
In this instance I can't count the cells as the they have to be confluent and are too packed to count individual cells. I was thinking it may be possible to take a picture of the whole well in both GFP, then CHERRY and use some software to determine the percentage of each. I will just have to scale it down to 96 well plates (not 12 well). Do you think this is possible?
It will be difficult to get enough resolution even at a 96 well level to be able to distinguish the % transfected when taking an image of the whole well. You can take several pictures of parts of the well and work from there, it should be close enough for most purposes. If you want a definitive answer, you can stain the nuclei of the cells with something like DAPI and use imageJ to count the number of nuclei, then c.f. with the number of GFP/cherry positive cells.
Incidentally, confluent cells don't transfect well, you need to have the cells in a log-phase growth or about 70% confluent for efficient transfection usually.