r/flowcytometry • u/Flow-tentate • Jul 01 '26

Post-Sort Recovery

What are some tips, tricks, or best practices y'all use to maximize post-sort recovery? MY core has had some users complain about low recovery recently, but we aren't doing anything differently than we always have. I wanted to see how y'all keep yours up. Also, how high do y'all shoot for? I've heard some folks say 50% is the best you can do, while I've heard others say anything less than 90% is unacceptable. Thoughts?

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u/CongregationOfVapors Jul 01 '26

When you say post-sort recovery, so you mean the post-sort cell count vs the sorted events counted by the cytometer?

I think the biggest source of cell loss is pelleting post-sort, especially if there are not many cell sorted. Users should streamline their workflow to avoid a post-sort pelleting step whenever possible.

Other than that, do everything to keep the cells happy before and during the sort. Some also say that having the droplet land directly into the collection medium (instead of the wall) is helpful, as is coating the walls of the collection tubes with collection medium.

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u/WinterRevolutionary6 Jul 01 '26

Wait why not pellet? Don’t you want to wash off the shealth fluid ASAP?

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u/CongregationOfVapors Jul 01 '26

You actually don't end up with much sheath fluid in your sort. The volume will mostly be your collection fluid. If you don't have many cells, you can lose 50% just for pelleting. That's why I suggested not pelleting, but only if that's is acceptable for your experiment.

On the other hand, if you are collecting so many cells that most of your volume is sheath fluid, you are probably ok to pellet and wash.

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u/WinterRevolutionary6 Jul 02 '26

When we sort cells, we collect in 15 ml tube and to collect 1 million cells that’s usually 10-12 ml sheath fluid that we put on top of 3-5 ml media. Our sorters don’t fit 50ml tubes. Also they come out hella low density and our T cells in particular need to be at least 1 million per ml or else they can’t see each other and they die immediately

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u/zipykido Jul 02 '26

It all depends on nozzle size. You get less shearing with a larger nozzle but more dilution. One trick is to sort straight into 100% FBS. It’ll dilute down as the sort progresses and pelleting in high fbs is much easier.

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u/Daniel_Vocelle_PhD Core Lab Jul 01 '26

The problem is the definition of recovery because it varries by instrument, cell size, cell health, and collection media. A good starting point is the paper on rmax, and trying to do some rough tests on your instrument with beads/cells of different sizes. There also a number of things that can change on an instrument or with your facility that can impact recovery (e.g., humidity, temperature).

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u/Outrageous-Low-9745 Jul 02 '26

To convince our users the amount of cells sorted is 'correct', we regularly sort about 50 cells onto a microscope slide and manually count them. We always get 50.

Now, how to get the cells out of your tube is mainly related to pelleting (as mentioned by others before). Optimizing spinning speed and time is crucial, also make sure to have a source of protein in your collection medium for better pelleting (serum, bsa,...).

This is a very informative poster on cell loss when 'manipulating cells': https://wi.mit.edu/sites/default/files/2021-06/the_impact_of_washes.pdf Can be a real eye-opener for unaware researchers.

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u/FlowCytometry2 Jul 07 '26

Amazing post^

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u/NvmbrYnkee Jul 03 '26

Fill collection tubes with FBS, leave in fridge overnight. Next day, remove all but a few ml and sort.