I would suggest using a biotinylated antibody followed by anti-biotin microbeads. If you are planning to go on to flow-based cell sorting, you can then stain the column-bound fraction with your other 3 antibodies plus streptavidin conjugated to a fourth fluorochrome to enable you to identify your target population.

Alternatively, if you only want to perform analytical flow cytometry on the macrophages, could you not just mince and digest your tissue of interest, clean up the sample and then stain with your antibodies of interest?  You wouldn't need the MACS step, saving you cells, time and money.  You could always counterstain with a fourth marker such as CD45 to look at the total leukocytes in the sample so that you can get a sense of what proportion are macrophages.

The labeling with the beads usually doesn't interfere with measuring the fluorescence on the flow cytometer; hence if you use the fluorochrome you would usually use for flow cytometric analysis you can just use this to attach the beads for separation and still would be able to see the label in flow cytometry. If you normally haven't got any problems with labeling all three markers together this will also not change when you add the beads. Only thing is you might need a different (higher) fluorchrome concentration to get the best MACS separation result. This you will need to test.

Hi Maria, we also provide a cell separation system (non magnetic - sieving technology). In case you are interested we can customize F4/80 separation beads for you. In case you are interested you can contact us/me at any time. Best wishes Michael

Hi Maria

DO you know how many macrophages you have? If you're working in small animals the number could be very small. I think you might be better using immunohistochemistry. THis would also have the advantage of giving you a situational context and you could do a limited amount of counter-staining (TNF or IL-10, for example).

Good Luck

G