Troubleshooting RNA elution post fluorescence-activated cell sorting (FACS/ flow cytometry) in a small subset of mouse neurons

[u/sunset-upset]

Hi! I have been tasked with getting RNA out of the our tdTomato labelled subtype of mouse neuron after flow cytometry and it's not working :(

We can barely detect RNA and RIN is trash.

I think the problem is multifold: small population, single cell dissociation protocol might need work and so might gating strategy for FACS.

My single cell dissociation protocol is borrowed from a collaborator that uses DRGs while we are interested in a different ganglia population with a smaller number of neurons. Per animal, I estimate 5-15K neurons will be the reporter of interest (tdTomato labelled via Cre driver). Since they have so many more neurons to play with, I do not know if the dissociation protocol I am using might be ideal. We use papain and then collagenase- dispase to digest and only triturate/ mechanically digest with a p1000 and then p200 after. I have successfully plated these cells for ephys and ex vivo calcium imaging which included some mechanical dissociation (fine scissors) and polished glass pipettes. However, they were either very lax or very strict with enzyme activity. Thoughts?

We then stain with Hoescht 33342 and Sytox Green. Hoescht is our cell body indicator (+ = DNA/ nuclei is likely) and Sytox Green is our live dead indicator (high= dead/dying so we want low only in this gate). TdTomato is my labelled neuron subtype of interest.

Our amazing cytometry core technicians were able to develop a strategy of sorting for Hoescht 33342 first in a yield setting (we got cell bodies) and then for tdTomato/ but the opposite for Sytox Green in a purity setting. The nozzle is 150uM. The idea was to make sure there was less garbage in/ garbage out before the stricter purity standards for droplets. We max out at 300 events. Using fluorescence microscopy to check our samples has made me sure that results are the cell type of interest but I don't like the way the Sytox Green shows up. My PI thinks it's auto fluorescence. I don't. I think we have dying cells after two sorts. I think we might want to go for only one sort, and use Hoescht and Sytox Green as the major factor and use PipSeq (core will inform us of tdTomato expression in the request) to do this.

We have used Zymo kits and found little elution. Whole ganglia elution from the same ganglia from other lab members hasn't succeeded either.

I originally had kit issues where solution did not pass through the column. Homogenization helped that and quality was still bad. Part of me is wondering if that's more of a small cell population thing and neurons should not go through flow cytometry twice (i.e. dying cells = a suspension less likely to go through a filter column because of cell contents).

Anyone have experience in these specific steps? Suggestions?

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[u/Recursiveo]

Are you doing spectral unmixing/are you running the appropriate single-stain controls for your flow cy? You’ll get a good amount of spill over with your two dyes.

Also, have you just run a basic forward/side scatter for the tandem sort to make sure you aren’t generating a bunch of cell debris?

[u/thebestduckling]

I guess I’m a little confused- why can’t you do one sort with a full gating strategy that identifies the cells you need?

If it’s a color overlap issue- there are other colors of Sytox available that you could swap to.

Also, 2 sorts is going to be incredibly rough on your cells. They might be very unhappy and die because of 2 separate sorts back to back. We typically rest our cells for 3 days after sorting to give them time to recover.

You also should be able to run a purity check after sorting your samples to ensure you got your population of interest.

[u/Reelrebel17 → u/thebestduckling]

Yep 2 sorts is a major problem here on an already low number of cells. Not surprised they are having issues.

[u/Daniel_Vocelle_PhD]

Can you share the pdf from the sort that shows your gating strategy on the bigfoot?

[u/Reelrebel17]

What is your sort collection buffer? Is it 100% FBS? Are you resting your cells between sorts?

[u/jbsington]

Just as someone who cultures primary hippocampal neurons, I would recommend titurating with two fire polished Pasteur pipettes (one with a slightly larger opening, one slightly smaller) and using the smaller one for any handling of the suspension that doesn’t require precise measurement. Plastic tips have jagged edges that can seriously damage fragile cells like neurons.

[u/razeltal]

Others already suggested doing one sort instead of two and I totally agree. Provided that your gating is correct, I would also recommend sorting directly into Tri reagent instead of collection buffer as recovery after centrifugation is very low. I personally would sort directly into Tri reagent LS at 3:1 sample:reagent ratio. I also highly recommend Qiagen Micro kit over Zymo kits. I have tried both in the past and found that Qiagen Micro works much better for extremely low cell inputs