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xinmimi77's Content

There have been 4 items by xinmimi77 (Search limited from 15-May 20)

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#153476 Gating of dendritic cells

Posted by xinmimi77 on 09 April 2013 - 08:32 AM in Flow Cytometry

Thank you!

#152920 Gating of dendritic cells

Posted by xinmimi77 on 26 March 2013 - 06:53 AM in Flow Cytometry

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.

#152880 Gating of dendritic cells

Posted by xinmimi77 on 25 March 2013 - 11:55 AM in Flow Cytometry

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.

#152872 Gating of dendritic cells

Posted by xinmimi77 on 25 March 2013 - 08:57 AM in Flow Cytometry

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.


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