Btw, the cells are not fixed during incubation

Incubation during 1 hour at 4°C as you did should definitely work, as in most cases 30 mn is actually enough. So the problem is somewhere else.

If cells viability was an issue (did you actually detect cells by FACS using FSC-A vs. SSC-A ? and did you use a viability dye ?), and if FACS buffer was prepared in-house, then one possibility (that occured to one of my students once) is a incorrect dilution of concentrated buffers (for example 10X PBS improperly diluted). This is very basic, but it's always worth double-checking that buffers were properly prepared.

If cells viability was not an issue, then you can double-check that you did have anti-mouse antibodies (and not anti-rat or anti-human) and that you used the propper FACS chanels to detect the antibody-conjugated fluorochromes.

All in all, it might help if you provided more details about your protocol (how did you collect your cells prior to staining for instance, or what fluorochromes you used, these kind of things).

I agree with Julien Verdier. 20 mins is more than sufficient for most surface binding antibodies. The problem seems to be elsewhere (buffers, antibodies or your cells expressing really low levels of the markers which them being RAW cells shouldn't happen). Julien has covered the various possibilities.

Thank you Julien Verdier and Julie Joseph ! I gues the problem might be cell death caused during cell detachment. I grow them in 24-well plate and when I tried to detach them before flow cytometry, instead of using a regular cell scraper (which is too large to fit in the well), I bent a pipet tip to serve as "scraper", which might be too harsh for the cells. Also I tried trypsin to detach the cells but RAW 264.7 cell line is really adherent so they hardly get off the plate. Next time I might try growing them in larger containers like cell culture dishes where cell scraper could be used.

Sebastian Schmitt

Hi Vincent

I work on RAW cells. I stain these cells frequently with MHC-II and CD-80/86 ab. Incubation of Ab for 30 min at 4 c is more than sufficient. I guess you need to standardise you cell harvesting protocol. You can use ice cold PBS for harvesting the cells if cell scrapping is not working in your case. Just add ice cold PBS in wells, pipette for 6-8 times. You will get all the cells in suspension.

Thanks

Pramod Gupta Thanks for hyour answer! This time I am incubating all the cell in 100mm dishes. I plan to use the cell scrapers in ice cold FACS buffer (485ml PBS++, 10ml FBS, 5ml P/S, 10uL EDTA), and do the washing and in falcon tubes or ependof tubes. I just worry that the cells might be adherent again during the staining step, so should I do sth to lift the cells again after that?

Vincent Fan once the cells are harvested, they won't adhere to the walls of eppendorf tubes. However these cells tend to form clumps after exposure to PBS, hence I would suggest that after each washing, dislodge the pellet and make single cell suspension before moving to the next step. In staining step, add your staining solution to the pellet and resuspend the cells in that solution followed by incubation at 4 C.

Pramod Gupta I just tried using the cell scrapers and I did get a lot of cells. However, when I ran flow, it showed that not a lot of cells are labeled with CD80 or MHC II ab, only like 30% ish labeled with each ab. Is that normal or sth might have gone wrong? I'm thinking maybe I have too many cells this time, cuz I have around 1E6 cells in each sample and I did 500x dilution for each ab.

You can try using EDTA only to detach your cells as it is less harsher than trypsin and do not cause degradation of cell surface proteins required for flowcytometer running!

Vincent Fan Resting macrophages don't have high expression of MHC-II or co-stimulatory molecules, therefore percentage labelling that you got looks correct, however you need to repeat your exp to find out 30 % labeling is correct. Further titartion of Ab may also be needed.

If you try to fix the cells before dyeing?

You have to standardize cell preparation and Ab incubation .Dead cell have to be excluded by suitable gating based on the scattering distributions. 100mm dished for this type of experiment is a waste. Also too many ceils will titer out your antibody. Try IFNg for inducing MHC II as positive control.