Lsr fortessa hts

Control and constricted MDA-MB-231 cells were cocultured with human NK cells sorted from peripheral blood using the NK cell isolation Kit (Miltenyi Biotec 130-092-657) at the ratio of 1:20, in triplicate for each condition. Before co-culturing, tumour cells were pretreated with 10 µg/ml mitomycin for 1 hr to stop proliferation, and were incubated and tagged with CFSE (Invitrogen CellTrace, C34570) at 1 µl/ml for 20 min. Twenty-four hours later, cells in each condition were recovered by trypsin and stained intracellularly with Granzyme B (Biolegend, AF647, clone GB11). Flow cytometry was performed by BD LSR Fortessa HTS and data were analysed using GraphPad Prism V9.

Two cytometers were used to collect data: BD Biosciences LSRFortessa and BD Biosciences LSRFortessa-HTS. Both were equipped with 405, 488, 561, and 620 nm lasers. Cells were run on the lowest flow rate possible. Voltage and filter sets used were as follows (two filter sets were used on the HTS instrument):
All individual experiments were performed with the same voltage set, and the fluorescence values reported are normalized to a within-experiment fluorescence baseline (unstressed cells), allowing for direct comparison between experiments taken on different instruments or with different voltage sets.
Unstressed cells were used to determine manual gates on forward and side scatter to isolate cells. Growth conditions (see above section) were such that no significant populations of dead cells were expected. In some experiments a sub-population of cells became highly fluorescent in the BV421 channel. These cells were ambiguously bright in the FITC (488) channel, meaning that they could not be confidently assigned to either strain; although recorded, these cells were excluded from the analysis computationally by threshold gating in the BV421 channel. The percentage of these cells of the total initially gated population was between 5% and 50%, and varied primarily with handling (no association with pH).