| | Re: Qauntifying surface receptors with FACS
The answer is simple. Firstly, you have to use same concentration of your primary Ab and isotype control Ab. Then you have to use a complete negative control for same cells along with isotype control to set the proper voltages on the machine. If you are using 5 log decade machine, like FACSAria or 4 log decade machine, like DAKO Cyan ADP, then you have to decide in advance after running your neg and isotype control where your cell population falls. Lets suppose you set voltages where your neg and iso controls appear just under 10e2 decade, then anything wchich appears beyond that threshold would be a positive outcome.
Now it depends on the expression of your surface marker, whether it is highly expressed or low, that determine your final dot plot. Also the fluorophore you used has its own quantum yield, that is how brightly is fluoresce. Therefore if you are getting low expression with FITC, try APC for the same marker to see whether there is any shift in expression (if you can afford to do that).