Staning macrophages for FACS - (Sep/05/2008 )

Hey all!

I have had some problems with the staning protocol, I have done both staining for FACS and for microscopy.
Instead of asking for a protocol, I want to see if you guys can find any errors in my own protocol.

1. Detach macrophages from wells by using 1xVersene for 30 minutes in 37 degrees celsius.
2. Spinn down the cells (1000rpm, 8 min, 4 degrees) and discard the supernatant (SN), save 100 ul of SN.
3. Resolve the pellet and add 100 ul of antibody solution (primary antibodie or antibody-cocktail, the antibodies are diluted in 1xPBS)
4. Incubation for 1 hour on ice. (dark if the antibodies are conjugated)
5. Add 1ml 1xPBS and centrifuge as before.
6. Discard supernatant
7. Resolve in either 500 ul FACS buffer or adding 100 ul of secondary antibodies
IF I USE SECONDARY ANTIBODIES
8. Incubate 1 hours
9. Wash with 1ml 1xPBS
10. Resuspend in 500 ul FACS buffer.
11. Analysis

If I need to do intracellular staining, then after step 1 Ill start with the fixation by using 200 ul of 4% Formaldehyd during 15 min at room temp. Then I wash the cells with 1xPBS, and add 0.1% Saponin for permab. I also dilute the antibodies with Saponin.

The problem is that for the secondary antibodies I dont get any staining, sometime I dont get any staining at all, this is new antibodies made for FACS. Another problem is that the LPS/IFN-g stimulated cells have a very different SSC/FSC profile which might be normal but looks weird hehe...

If you have a great protocol which works for both intracellular and extracellular staining on macrophages, please post it.

Best regards
Roham

FACS staining macrophages should be easy - is your staining for other cells (with different antibodies) working at all?
Clare