In my opinion SCC is not a good measure to use as a gating strategy. There will be considerable variation in SCC even for a single cell type.

Was there not enough detail on FSC-H x FSC-W or FSC-A x FSC-H/W to remove debris?

Ideally you would run a control that had some previously isolated cells to set a size filter/ threshold.

Secondly, what are you CD marker gates set on? Are these set to negative/ positive controls? It seems as though you have two separate populations..

Looking at your plots, I am not sure that you have successfully removed all debri based on FSCA/FSCW and SSCA/SSCW. The population of interest looks separate in the "contralateral hemisphere" plot (it is the ellipse near 1.5-2000 000 on the X axis). To me it looks as if the same cells are shifted closer to the axis origin on the "ischaemic" hemisphere FSC/SSC, which could be a biological effect (e.g. are they dead and fragmented due to ischaemia?). But you are certainly clipping quite a large amount of debri in your initial gate, which is probably affecting how the subsequent gates look (debri stick to antibodies non-specifically).

To work out where your cells of interest sit, try skipping the first four gates; start by gating only on FSC/7ADD negative cells, followed by the markers of interest (CD45 and CD11b or CD11c), and finally back-gate on the FSC/SSC plot (i.e. overlay this plot on top of the FSC/SSC plot). This should give you some idea of where the viable double positive population is (if there is one) and enable you to set the first gate correctly.

I am unfortunately not sure what microglia should look like because I mainly work with lymphocytes. But from having glanced at colleagues' data on innate immune cells like monocytes and microglia, I got the impression they may not always have such distinct populations as lymphocytes would (e.g. CD4+CD8- etc). Rather they may express more or less of certain markers. Which means you may probably need to include some FMO controls to work out where your positive and negative CD45 and CD11b/c gates should be (if your cells are very precious, you can do this on a control subject, and provided your settings are reproducible, you may not have to do it for every experiment).

https://www.rug.nl/research/portal/files/41613182/Chapter_5.pdf

Maybe this will aid you? This is the way we do this in our facility (see plots).

Hello Johan Teunis ,

Thank you for sharing your protocole ! I have a question about it.

In your protocol you note:

"12. Resuspend the cell pellet in 1 mL 60% Percoll using 1 mL pipette tip.

13. Add 14 mL of 60% Percoll solution (per 50 mL tube) to the suspension

and homogenize.

14. Carefully place 14 mL of 30% Percoll on top of the 60% Percoll layer.

15. Carefully layer 3 mL PBS on top of the 30% Percoll. Centrifuge for 25

min, 800 g (acceleration 4; brake 0)

16. Collect the 60%-30% interphase with a pre-wetted (with 1 mL iMed+)

glass pipette and transfer to a 50 mL tube containing iMed+. "

It must be understood that the cells in the 60% gradient, located at the bottom of the tube, rise towards the 30% gradient, located at the top of the tube, during centrifugation? Is it due to the density that the cells react this way? Why not mix them with the 30% gradient to lower them to 60%?

Sincerely

Good question Lucas Le Roy! Indeed the density of the microglia is less then the 60% and more then 30% and so it moves upwards! You could also mix the pallet with 30% and put it on top of the 60% but this will create an additional step since you have to pipet the 30% with cells again. But other then that it should work I guess!

Hello,

Thank you for your answer. It's an interesting way to make a percoll gradient !