FACS and isotypes - (Jun/22/2006 )

Hi, anyone do alot of FACS? My directly conjugated isotype is so far right of my no stain.
This is all human continous cell lines. What I am not sure of is whether this is masking the
actual test antibody result, which is shifted to the right of this isotype, but just slightly!
I just got an Fcy-binding inhibitor, and I know that CD16/32 blocking is common in mice
work, but apparently not so in human work. I tried the inihibitor and it did shift my isotype,
but unfortuantely it also shifted my test antibody as well, so now I everything is overlapping
the no stain?!?!?!?

When you say overlapping, do you mean that the plots can be superimposed and there's not part of the curve to the right hand side? In this case you have no detectable expression and your previous staining was down to Fc receptors. If there is some rightward shift of the staining then you have some low level or a subset of cells expressing. Use the histogram statistics and some marker gates to try and work out the m.f.i. of expression and % of cell expressing.

Using non-conjugated primary abs (and an isotype in parallel) plus a FITC/PE-conjugated ab may give you more sensitive detection. What cells are you using and what cell surface protein are you staining for?

All the best,
Ceri

So when I use PE-conjugated Ab and appropriate isotype I have no stain at 10-1, then the isotype is shifted to the right of that, and the test Ab to the right of the isotype (slightly, but a clear shift). Some people had commented on the fact that my isotype and my nostain should be more equivalent, but there is no overlap with them, but I do see a difference in the isotype and test antibody, which I thought is what is relevant. BUT, yes, when I use the human Fc inhibitor, the isotype histogram overlaps/superimposes over the nostain hitogram, and basically so to does my test antibody, there is a very tiny shift for the test antibody, but preportionally not the same (between isotype and test antibody), as the difference I seen with the Fc inhibitor.

I thought that the PE-conjugated antibodies would be more sensitive? Are you saying that unconjugated actually are more sensitive? Why would that be? I have those antibodies, so I could do that test,

SO my dilema is what is truly accurate, doing the PE-staining with appropriate isotypes, or doing PE-staining after pre-treatment with Fc-inhibitor, with the appropriate isotypes??

It sounds like you have some binding because of Fc receptors if your isotype and non-stained sample don't superimpose and don't overlap. I guess having some labelled ab you probably will get some shift, that's the reason for using an isotype. If the PE-conjugated test ab gives more staining than the PE-isotype and they're from the same company and used at the same concentration these results are valid even if you don't use the Fc inhibitor.

Then it is worrying if the Fc inhibitor blocks this result, although you still get some staining. Have you tried titrating the Fc inhibitor down to see if you can still have no staining with the isotype and more staining with the test ab. Do you use any other blocking agents in your FACS staining protocol?

The reason I said using unconjugated abs should be more sensitive is more than one secondary can bind to the primary so you should get an amplification of the signal (like in westerns or ELISAs) so using a primary, then a biotinylated anti-species of the primary, then streptavidin-PE should be more sensitive again. This is in theory as I did get better staining with a unconjugated versus a conjugated but they weren't the same antibody. The only downside is you need extra controls (2ndary alone, isotype plus secondary etc), it could be more non-specific, and you need longer for the extra wash steps and staining steps before going on the FACS.

Regarding your results not sure what you should believe. Sounds like you have staining in both methods it just might be less in the second. Try using good positive and negative control cell lines with both methods and see which results seem more believable.

All the best,
Ceri