Pe mouse anti human cd9

OVCAR4 cells were cocultured with NK-92 or NKL cells at a 1:1 ratio
in U-bottom 96 well plates for 24h at 37°C. Cocultured cells were
harvested and washed with serum-free RPMI-1640 medium. Cells (2 ×
10⁶) were stained in 90 μL of serum-free RPMI-1640
medium with 5 μL of PE mouse anti-human CD9 (BD PharMingen, clone
M-L13) and 5 μL of APC anti-human CD45 antibody (BioLegend, clone
HI30) for 30min on ice. The cells were washed with serum-free RPMI-1640
medium and stained with DAPI (BD PharMingen, 0.1 μg/mL) in 1mL of
serum-free RPMI-1640 medium for 10min at RT, quenched and washed with
complete RPMI-1640 media (supplemented with 10% FBS, 2 mM L-glutamine and 1%
pen strep). Cells were resuspended in 1mL complete RPMI-1640 media and
filtered before sorting. CD9+ and CD9− NK cells were collected in
FBS. Cells grown in monoculture were used as controls.
Cells were sorted on a Sony SH800 cell sorter, according to the
following steps: i) FSC- versus FSC-A for singlets ii) DAPI for live cells
iii) CD45 for NK cells and iv) CD9 versus CD45 to collect CD9+ and
CD9− NK cells.