I used to gate NK (CD56) and CD3+ cell in the same sample. According to previous literature, the NK cells are only 5-28% of the lymphocyte population in peripheral blood. However, I found that my sample have around 50% NK cells, Which are not normal.
(Biburger M, Nimmerjahn F. Immunol Lett. 2012 Mar 30;143(1):53-9)
Looking at the paper (Biburger M, Nimmerjahn F. Immunol Lett. 2012 Mar 30;143(1):53-9), what it says is that NK cells are not really "sticky" but monocytes are and bind via Fc receptors to NK antibody. Thus, the NK cell gate is contaminated.
The high % of NK cells in my sample is due to above mention reason? And would it be helpful to use some Fc blocker ?
or there may be some other reasons ???
Thanks.
One very simple way to find out if your excessive percentage of NK cells is due to nonspecific binding to monocytes would be to check if you have an NK signal (CD56) in the monocyte population. For this you do not even need a monocyte marker, as you can discriminate lymphoid and myeloid populations on an FSC/SSC bivariate plot. By overlaying your gated NK population on this FSC/SSC plot you should only see the overlaid population in the lymphoid population, not in the myeloid population. If you do see a CD56 signal in the myeloid population, then using FcR blocking reagents might be a solution indeed.
Other reasons explaining your result could be listed but for this it would help to visualize your gating strategy.
Hello Waqas, the choice of conjugates here could be an issue - what fluorophore(s) are your CD3 and CD56 conjugated with?
Do you use any blockage procedure?
Julien Verdier Thanks. okay i will try to used lymphoid and myeloid populations on bivariate plot of FSC/SSC.
Martin Waterfall I used Cd3(Percp) and CD56(Pacific blue). Before running the sample, i set the compensation with BD beads, and the separation of both these Abs are perfect and didn't see any overly with each others.
No, i didn't add any blocking reagent.
As reported, in some data sheets, cyanine conjugates like Cy5 have a specific affinity for Fc receptors, giving selective co-labelling of FC positive cells. I've not seen reports for PerCP but PB conjugates can bind Fc. Try blocking with 1-2% antibody host(eg mouse) serum or something like Human TruStain FcX. Alternatively choose a non-binding conjugate PE, APC.
Martin Waterfall Thanks. yes i am planing to add blocking reagent, I hope that will work.
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