We are working on a protocol for the isolation and characterization of macrophages from human tissues (skin). We use the following antibodies, CD40, CD80, CD163 and CD207 (all surface markers) to discriminate between M1 and M2. In addition we use CD68 to gate all our macrophages.
However, CD68 requires fixation and permeabilisation prior to staining as this is an intracellular marker. The other antibodies do not require fixation and permeabilisation. Which option is best to stain these markers.
Option 1: Stain cells with surface markers, wash, fix, permeabilise and then incubate with CD68. (Our concern: Does the fixation and permeabilisation step affect the other antibodies, i.e. quenching of the probe?)
Or Option 2: Fix the cells, then stain for the surface markers, proceed to permeabilisation and the perform CD68 staining. (Our concern: Does the fixation affect the epitope of the surface marker which may results in less efficient binding of our antibodies)
Hope you can share your experiences on this matter.
Marcel
I would go with option 1. This is widely used when staining intracellular/surface markers on immune cells. Fix/Perm should not affect surface staining. Seems BD's Cytofix/Cytoperm would work with CD68.
http://www.bdbiosciences.com/us/applications/research/intracellular-flow/intracellular-antibodies-and-isotype-controls/anti-human-antibodies/purified-mouse-anti-human-cd68-y182a/p/556059
Haven't done this for tissue macrophages, just suggestion. I would start with Fc block, then surface staining, then fixable viability dye, fixation with PFA containing buffer, permeabilization with saponin- containing buffer, blocking again, and then intracellular staining for CD68. For macrophages I will avoid too many colors as these are rather autofluorescent cells and will require too many controls (and too many cells for fluorescent-minus one controls in particular). And first will try to compare surface stained alone (with viability dye as a must) with surface stained then fixable viability stained /fixed cells to make sure surface antigens are not altered by fixation. If you are going to use ficoll gradient to get rid of dead cells or magnetic kits for dead cell removal and maybe macrophages magnetic-enrichment kits, viability dye may be skipped. If you are going to enrich macrophages by adhesion there will be many dead cells after scrapping and viability dye is a must. In general I highly recommend dead cell removal and pre-enrichment prior to staining of macrophages. I would also very likely add DNAse to the staining buffer to reduce clotting.
Sounds like a consensus to me. Ultimately, the only sure way to answer is to do it. Good luck!
I have done surface+intracellular staining in the same tube plenty of times with option 1 only. I don't have experience with option 2 to answer if it works similarly or not, but I assume that with option 2, you might risk non-specific binding of the surface antibodies onto newly revealed intracellular epitopes after fixation/permeabilization (depends on the antibody of course, how its specificity is tested by the company etc).
So, my recommendation is with option 1 to be on the safe side.
By the way, since you are considering intracellular staining, you might be interested in staining for various cytokines for instance? Or signaling molecules? This would help to discriminate your macrophages further in the spectrum of M1 to M2 subsets.
Here you can read a nice review regarding markers of various macrophage subsets.
https://www.hindawi.com/journals/mi/2015/816460/
I also attach a useful table from BioRad to start with.