Gating of dendritic cells - (Mar/25/2013 )

I have a question regarding FACS analysis of dendritic cells. I understand that aggregates need to be excluded during analysis. When we prepare cells for FACS analysis, we always try to disaggregate dendritic cells, which are notoriously sticky (e.g. with the addition of an anti-clumping agent). But unfortunately some doublets still show up on our FACS plots. We have routinely excluded the doublets in our analysis.

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.

Xin

I've got a question on that. Are you really sure it's OK to knowingly include doublets in your analysis ? Because doublets should, as you also stated, normally always be excluded if possible. Doublet means you have two cells sticking together but the Flow cytometer recognizes it as one fluorescence signal, so it's basically distorting your data (especially if the amount of doublets is not equal between the two groups, it's not easy to compare them).
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.

Thank you for your reply. I do not want to include the doublets either. However, I also don't feel comfortable to exclude them because they are indeed CD11b+CD11c+ cells. (I am also wondering if our treatment has made the dendritic cells less sticky/adherent, thus leading to fewer of them stuck together than the control group. That would be an interesting lead to follow up on if true.)

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.

Thank you.

xinmimi77 on Mon Mar 25 19:55:52 2013 said:

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).

Thank you Tabaluga. We've got more than that, like 15-20%. When you use Accutase, do you leave it there all the time (e.g. during FACS run)? If so, what percentage of Accutase did you use? Thanks.

No, I don't leave the Accutase there all the time - leaving it on the cells for more than some minutes destroys them. When I harvest my cells (suspension cell culture) I centrifuge and take off the medium, then I resuspend them in 500 ul Accutase and pipet it up and down till the solution looks homogeneous, which might take 2 minutes. Then I stain and before measuring I pass them through a cell filter.

EDIT: I forgot to add in the above post that I add PBS to the Accutase-resuspended cells and centrifuge (wash) them once before I start with the actual staining.

Thank you!