Hello,
Our lab has been dissociating solid colon/small intestinal tumors from mice. We use a fairly standard dissociation protocol that has been looked at by 10X. After we get the single cell suspension, we sort them using a FACSAria cell sorter.
Despite sorting, when we check the viability we still see only 60-80% viability.
Does anyone get higher viability post sorting?
What is the percentage viability post-sorting needed to run single cell RNA sequencing?
Any information will be appreciated!
Hello Apryl Saunders
Post-sorting cell viability is always a problem. I would suggest you use serum or protein in your buffers as this will increase viability of your cells. Use 1-3% BSA or FBS in buffers. It will help keep the cells healthy and viable without making them too sticky. You can also coat your collection tubes with protein (1% BSA) prior to sorting as this will help reduce the chance of sorted cells from sticking to the sides of the tube. This will increase sort yield and cellular recovery. If you are using media, you can use a concentrated media that has a high FBS content (~20%) to help cells recover from the sorting process.
Also, use the right temperature. Keep cells on ice but not for too long. Keeping the cells on ice can preserve viability. However, cold temperature can also prevent cells from repairing sort-induced damage. So, the longer the sort, the more damage done to the cells which cannot be repaired.
To maximize the amount of useful sequencing data in single-cell RNA sequencing, the fraction of dead cells must be minimized. Recommended cell viability should exceed 90%, with a minimum up to 70% cell viability.
I hope this will help.
Best.
It is possible you can avoid sorting entirely? Probably depends on how many cells you are capturing, but if you don't mind a little bit of contamination with non-tumor stromal cells (which are usually ease to detect later in bioinformatic) and depending on your model could even use the same marker you are using for FAC sort. If your tumor samples after dissociation look quite enriched in tumor cells maybe would be a good idea.
Alternatively, in a completely different way to look at the problem, and if Malcolm answer doesn't work for you, if you are targeting a really specific population in your tumor that need to be enriched you could try an intermediate step generating organoids. Organoids have prove in a number of models that resemble quite good the internal heterogeneity of the original tumors and they have the advantage that are really ease to dissociate with a higher cell viability than direct tumor dissociation.
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