I've seen this for awhile and just assume it's normal as I add in Fc block to prevent extra background but thought I would see if others also see what I am seeing. Maybe others dont use isotypes for every condition in their assay though.... If you look at the fluorescent signal for the isotype stained cells from unstimulated and then for example at TLR ligand stimulated cells, I'm noticing if you overlay the histograms the isotype controls are definitely different with the TLR ligand stimulated cells giving a higher MFI. So when I add the receptor antibody I will subtract the respective isotype control MFIs so it's normalized. Is that the best way or I guess I could calculate a fold difference over isotype? The receptor is not highly expressed so an isotype is essential. I've seen this for B cells and monocytes so far. Thanks for any feedback.
The mechanism might be you a regulation of Fc receptors - most people block only with the common Fc block (which blocks in mouse only Fc gamma receptor 2b and 3). Fc gamma R 4 is also able to bind a lot of the commonly used antibodies for staining (especially if you use antibodies generated in a mouse host). You u might want to think about blocking this receptor as well (by 9E9) or to use a de-glycosylated variant of your staining antibody (which would not longer bind to FcRs) ...
But to better help U we would need more details...
Thanks for the replies. I've attached an example histogram overlay for why I don't think I can use just unstimulated versus with CpG without isotypes or else I would have thought these cells actually expressed the receptor but when compared to respective isotypes there is definitely no difference! I'll see what that paper says you mentioned Dominic but hope some FACS expert can help out too.
The Fc block I'm using is from ebioscience so they mention the FcRs but dont say whether their inhibitor blocks all of them. I'm using a mouse IgG1 which is normally better for less binding compared to mIgG2a for example. I guess no matter what though if the 2 antibodies are staining similarly that should mean there is no expression on these cells. Now that I think of it human cells dont have FcgRIV as from when I used to work on FcRs I thought that was a mouse receptor only but could be wrong....
So I guess I'll just keep researching what's best to do and for now based on the isotype vs actual antibody I would conclude these cells do not express the receptor I'm looking at but I could see people saying a cell could express any receptor upon stimulation if they hadn't put an isotype control on both sets of cells... oh and I did lots of gating to gate out doublets, dead cells and other cells via surface markers
Sorry deanna - i didn't realize that you were talking about human samples. You're right, that there is no receptor called Fc gamma RIV, but the closely related Fc gamma RIIIA. It's really a pitty that ebioscience doesn't tell you which receptors their Fc-Block is blocking...
You're also right, that your mouse IgG1 is a good isotype. One should also take into consideration, that isotype-controls often have a higher concentration when delivered...
Is the isotype-staining also higher than the FMO control (so without any antibody in this channel) - if this is not the case maybe your cells are just more autoflourescent when stimulated - the may also change other markers that could lead to a bigger spillover in your receptor-detecting channel (or just "new" cells are now in your gates).
Normally all this things mean that your receptor is propably not there, but it's still possible that you just don't see it.
Is there a possibility to enhance your signal (for example by Biotin/SA or by any secondary antibody)? Because I would think that you might need a better resolution for solving that problem.
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