I am staining whole blood with antibodies like CD3, CD56 and NKp30 to assesses the expression of NKp30 on NK cells. I am getting increased percentage of NKp30 on NK cells in one group than in another, however, the median fluorescence intensity I am getting is reverse. So, please suggests what could be the reason for this?
Hi subrat
Have you titrate your antibodies? I think you don't saturate all your epitopes (you need more antibodies in your condition). When the number of positive cells for NKp30 increase the quantity of antibody available for each cell decrease accordingly. Thus your median of fluorescence is also decreased.
Best regards.
Its common for MFI to go down on activation. This is certainly true for CD3
You should titrate the antibodies against your cells and do you use any fc receptor blocker? If not sometimes that can also become important.
as other collegues wrote before , may be you should titres your antibodies. in my opinion you have to find the corrected concentrecion of antibodies necessary to cover all the epitopes.
Try using more concentrated antibodies for labelling, let's see if you have different outcomes form the present ones.
1) Titration ABs
2) Maybe you are gating the positive cells in a wrong way, including also negative cells. Use a FMO to better understand where to put your gates.
3) Use Geometric mean instead od MFI. It should give you a more accurate result.
regards
Hi Subrat.
You have to titrate your antibodies, in order to get the adequate concentration to saturate all your epitopes. (This is in most cases the reason to get results like yours). On the other hand, there are some activation molecules that are sheared off after stimulation ( I do not about NKp30 molecule, but should be a possibility)
Good luck with your next experiments
Sergio
Most of the people arguing with titration... of course this should be done, but besides this I think it is really worth thinking also about gating, blocking and especially like Lesley Ann Bergmeier mentioned: "Its common for MFI to go down on activation." I see this upon activation also on CD4 cells (CD4 epitopes were downregulated).
Downregulation of TCR in the literature:
http://www.nature.com/icb/journal/v84/n2/full/icb200620a.html
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