Anti gm1

Flow cytometry staining of MDA-MB-231, SUM159, and their respective ST8SIA1-knockout (KO) cells was performed using a previously described method (6 (link)). In summary, cells (~1×10⁶) were incubated with allophycocyanin (APC)-conjugated anti-GD2 antibody (BioLegend; San Diego, CA) for 30 minutes on ice in the dark. For measuring the expression of ceramide, GM1 and GM3 the cells were stained using anti-ceramide (Enzo Life Sciences, Farmingdale, NY) or anti-GM1 (Abcam, Cambridge, MA) or anti-GM3 (Cosmo Bio USA, Carlsbad, CA) unconjugated antibodies. After washes, the cells were incubated with APC conjugated anti-mouse or anti-rabbit IgG secondary antibody. The cells were washed once with FACS buffer containing 4′, 6-diamino-2-phenylindole (DAPI; 1 μg/mL). The cells were analyzed by an LSR II (BD; Franklin Lakes, NJ) or Galios (Beckman Coulter; Brea, CA) flow cytometers. Cell sorting was performed on the FACSAria II (BD) as described before(10 (link)).