FACS Controls for eGFP Sorting - (Feb/01/2010 )
I hope you can help, I'm not getting many straight answers and I'm new to FACS sorting.
I have eGFP (endogenously expressing) cells that I would like to sort off as a pure population from the rest. To set up the FACS machine, I understand I will require controls to set up the 'gates' (ie. whats included as 'green' or not). I would like to use beads to calibrate as the cell I am using are precious and difficult to obtain.
For this, what controls will I require?
1) An eGFP positive bead for identifying the fluorescence, and a negative for setting up voltages
2) Only a negative for setting up voltages, since I wont be required to setup compensation given that there is only one fluor.
3) Something else I don't even know about??
Thank you very much!
You either will have GFP signal or you won't, the question is how much signal you'll get. Controls will be more necessary if your cells autofluoresce (this is true for macrophages) making subtle GFP expression very difficult to separate from natural cell fluorescence. Another control to consider is to evaluate GFP expression intracellularly. Here, you're not looking for GFP fluorescence but for the actual protein expression.