Gating of dendritic cells
Posted 25 March 2013 - 08:57 AM
However, we recently noticed that our data on CD11c+CD11b+ cells would change dramatically with or without the inclusion of doublets. When doublets are excluded, the treatment group has more CD11c+CD11b+ cells than the control group. But when the doublets are included, the result is completely opposite, with the control higher than the treatment group.
I have tried to google for commonly accepted gating strategies for dendritic cells, but the only answer I have got is to exclude doublets. But as you can see, it may not be appropriate to exclude them in our case.
I wonder if you have any suggestions on this. I would appreciate your help.
Posted 25 March 2013 - 11:35 AM
I don't know what exactly you are investigating, of course, but I would rather trust the result without doublets than the one with doublets.
Posted 25 March 2013 - 11:55 AM
You are correct that the flow cytometer doesn't read correctly with non-single cells. But I don't want to simply exclude cells just because they like to stick together (and dendritic cells "love" to stick with each other). Do you happen to have a reliable protocol to reduce doublets? We have tried DNase I, EDTA, and Invitrogen's anti-clumping agent (don't know what's actually in it), but some doublets still remain.
Posted 25 March 2013 - 02:48 PM
Do you happen to have a reliable protocol to reduce doublets? We have tried DNase I, EDTA, and Invitrogen's anti-clumping agent (don't know what's actually in it), but some doublets still remain.
How many percent of cells are doublets in your measurements ? Unfortunately I don't have a good protocol. I simply dissociate my cells (but they're completely other cells than yours) with Accutase and pass them through a cell filter before measuring. Still, I've got at least 2-3 % doublets (sometimes more).
Posted 26 March 2013 - 06:53 AM
Posted 27 March 2013 - 09:44 AM
Posted 09 April 2013 - 08:30 AM