FACS Binding Assay for Cell Lines

FACS binding experiments were performed with CHP-134 wild-type and CRISPR knockout clone cells as described previously [22 (link)]. Cells were suspended in fresh RPMI 10% FBS medium. The cell count was adjusted to 10⁶ live cells/mL. Test Abs and controls were added (10 μg/mL) to cells in tubes and incubated at ambient temperature for 45 min while mixing by mechanically rotating the tubes. The cells were washed with RPMI 10% FBS, then resuspended in the same medium containing secondary goat anti-mouse IgG AlexFluor 647 (Thermo Fisher Scientific). After 30 min incubation with mixing, the cells were washed once with Dulbecco’s phosphate buffered saline (DPBS) without Mg²⁺ or Ca²⁺ salts and suspended in DPBS containing 0.5% (volume/volume) formaldehyde for 10 min at room temperature. Binding was analyzed using a LSR Fortessa Flow Cytometer (BD Biosciences, San Jose, CA). FlowJo (TreeStar, Woodburn, OR) was used for data analysis.