Hi all
We are doing some scRNAseq from human tissues and getting poor survival (as by TrypanBlue) after FACS sorting, even if we select for DAPI- in FACS. We suspect FACS is stressing/killing cells, what results in few cells rescued after sequencing and unreliable transcriptomic profile. We are considering a double filter strategy to isolate single cells, getting rid of debris, and loading directly into 10x without FACS sorting. Do some of you have experience with this approach? Thank you!
Hi Victor,
FACS sorting doesn't kill the cells, or it will never lower the viability of the cells. Unless you are maintaining every thing properly.
So try few things or modify if you are not doing it yet.
- Process the cells for sorting at 4 degree.
- Please check the sheath fluid pH.
- Take some media (0.5ml) in collection tube.(the tube in which you acquiring the cells after sorting. As the cells will be in stressed condition due to passing trough the lasers)
- Make sure that you platting immediately after the sorting without delay.
- Do the doublet discrimination by SSC height vs SSC area.
- Use Hoechst 33342 stain to get the live cells only.
All the best.
Utsav
It depends on the cells. Dendritic cells are more fragile than T-cells for example.
In all cases, you should minimize sorting time by enriching your population by either positive or negative selection with bead coated antibodies and a magnet (MACS or Dynal beads are examples). Once you have a 50-80% pure population sorting time can be as low as 10-15min. Of course collect the cells in FBS rich media to help them recover from the stress.
Thank you all. The beads are a good idea indeed, but we are currently interested in all cell types so using no antibodies (just DAPI- selection in FACS). We are collecting the cells in PBS containing 2% BSA.
I am following this discussion with interest as I am interested in scRNAseq and do not have access to a FACS. Have you made any progress??