The day of the assay (after effectors are plated) is long because of the 5 h incubation. Be sure to start early in the day.
During the setup of the assay, to avoid shocking the cells, pre-warm media and pipette gently, up until the staining steps. After the 5 h incubation, the aim is to stop all reactions, so pre-cool centrifuges and staining buffers to 4°C. Once cells are fixed, this cooling is no longer necessary.
The manufacturer recommend live/dead staining in PBS, but a side-by-side comparison of staining in PBS or flow buffer revealed no difference in performance of the NIR dye in our hands.
The manufacturer recommends washing in perm buffer after staining, but a side-by-side comparison of washing in perm buffer or flow buffer revealed no difference in performance of the staining in our hands.
When working with tissue samples, blocking prior to staining is required; filtering through a 70μm filter prior to running through a flow cytometer is required; and counting beads can be beneficial for accurate assessment of cell number.