Qauntifying surface receptors with FACS

[jackthehammerrr]

How does one quantify surface receptors with FACS? How does one even know if he/she has the receptors if comparing to the isotype control say at 5% gating, there's say 7% gating? (ie. a tiny shift to the right compared to isotype on the histogram when overlayed)

this has given me so much trouble. thank you guys in advance!!

[mykalachi1]

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).

[brettsportler]

Dear Jack,

I probably read this to late, but make sure that you match your specific mAB and your Isotype control according to their protein content!! Do not just dilute it the same way as your specific antibody as protein contents may vary extremely between the two ABs and affect your staining (for example, I am using a mAB specific to CD15, Protein content is 1,5yg/ml; the appropriate isotype control has a protein content of 50yg/ml! That is I dilute my isotype 1:33 as compared to the specific AB).

Hope this helps, regards

Björn