How would you set up a cell sort to get a pure, viable population for downstream culture?

Why

A sorter has to deliver cells that are pure, alive and still functional. The Cossarizza guidelines give the practical steps. Sample buffers such as calcium and magnesium-free PBS or HBSS with HEPES and 1 to 2 percent protein do better than bicarbonate media, because pressure in the sample chamber can reach several atmospheres.

The nozzle should be at least five to six times the cell diameter, and larger nozzles run at lower pressure, which suits fragile cells. Purity modes reject drops where a wanted cell sits near an unwanted one, at some cost in yield. A sticky sample raises the abort rate, a sign that cells are not arriving singly.

If the sorted negative fraction is very pure but the positive one is not, undetected doublets are the usual cause. Sterility needs cleaned fluidics and sheath, and a check that the sort stream is aimed correctly.

How to work it through

1. Filter the sample just before sorting and keep it cold in HEPES-buffered, protein-containing buffer.
2. Choose a nozzle at least five to six times the cell diameter and set the drop delay with beads.
3. Gate on scatter, singlets by area against width, live cells by viability dye, then the target markers.
4. Run a purity mode at an event rate the sorter handles without heavy aborts.
5. Collect into tubes precoated and partly filled with culture medium.
6. Re-run a small aliquot of the sorted cells to measure purity and viability, and count recovery.

Where the answer stops holding

Sort pressure and shear can change gene expression and activation state even when viability looks fine, so sensitive readouts may need an unsorted control processed alike. Very rare targets may need a fast enrichment sort followed by a purity sort. Antibody binding during sorting can itself signal through the receptor, which matters for functional assays.