Question about FCM dual labeling experimental design - (Dec/12/2007 )

Hi Guys,

I'm designing my first immunfluorescence experiment to verify the efficacy of my cell separation by magnetic sorting. I'm attempting to enrich an epithelial population from a mixed population of epithelial/immune cells. I'm utilizing two antibodies to do this. One which is a epithelial specific FITC conjugated antibody and another with is a pan-leukocyte PE conjugated antibody. The thing that I'm confused about is how I should be setting up my appropriate controls.

If I was planning on examining three populations of cells: pre-magnetic sort total cell fraction, post magnetic sort enriched epithelial population (positively selected), and post magnetic sort non-selected (everything else) for both these markers would I need to include 6 stained samples for each population for the FACS analysis?

1) Unstained
2) Isotype FITC
3) Isotype PE
4) Epithelial speciifc FITC
5) Leukocyte specific PE
6) Epithelial FITC + Leukocyte PE

Would that be necessary for each of the populations im examining? Or would 1-5 only be necessary for one sample (total fraction) to calibrate the instrument? Flow sorting would likely have been easier but we dont have a flow facility up and running yet and the MACS platform was readily available.

Thanks!

Fot the FACS analyis, you only have to include three samples for the calibration of the flow cytometer .
- unstained cells
- PE staining
- FITC staining

I yould use the population where you expect the best and strongest labeling.

Then for each population I would do a dual labeling with specific FITC and PE antibodies, and I would add a control of specificity of the antibodies by labeling the positive populations with the isotype control