I'm trying to analyse the expression of CD11b and CD62l on unstimulated and activated neutrophils. the CD62L works as expected, but the CD11b isn't doing what I'd expect - it works too well in an ironic sense!
What I'd expect is that the CD11b would increase on activation, but I found no differences between activated and non activated, and even after reducing the amount of antibody I was using, the amount of positive staining was still too high.
I'm wondering if anyone has had this problem as CD11b and CD62l are commonly used in activated neutrophils and I can't seem to find any graphs to check my flow data against
Hi Sophie. CD11b (or integrin aM), is upregulated on activated leukocytes.
I suspect that you are not using endotoxin-free regents for your neutrophil isolation.
I don't know about L-selectin (CD621I), but neutrophils will activate even if you think about endotoxin (joke).
Hi Sophie,
It could be endotoxin in your reagents as Steingrimur has suggested, although I would expect to see some modulation of CD62L as well if that were the case.
Have you titrated your anti-CD11b antibody? Also, what fluorochrome are you using it on and what cytometer? Have you done any isotype controls? Or F-1 controls? There's lots of reasons for high fluorescence that might be non-specific but without more details on your experiments it's not possible to say.
You also must consider the culture time, the purification and cell viability. Neutrophils express basal levels of CD11b, you can analize the change in MFI after activation.
Hi all,
thanks for your comments, I actually diluted the cd11b and it seems to have worked, and yes I know neutrophils ability to activate themselves for no apparent reason, makes them hilarious to work with!
the cd11b is a PE and the cd62l is an APC. I think I've been told that PE is quite a bright fluorochrome so this may be the reason the dilution has worked a tad better.
thanks for the tips
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