Biotinylated l pha

Cells were washed with PBS containing 0.5% (w/v) BSA (PBS/BSA). All staining was carried out in this buffer. For directly labeled antibodies and lectins, cells were then washed two times with cold PBS/BSA buffer, resuspended in this buffer, and analyzed by flow cytometry. For indirect detections, cells were washed once with PBS/BSA before the addition of the appropriate secondary detection agent. Cells were then kept on ice for 20 min, washed two times with buffer, and resuspended for flow cytometry. For PD-L1 detection in mouse cancer cells, APC anti-mouse CD274 (B7-H1, PD-L1) antibody (Biolegend, 124311, RRID:AB_10612935) was used at a 1:100 dilution. For PD-L1 detection in human cancer cells, PE anti-human CD274 (B7-H1, PD-L1) antibody (Biolegend, 329706, RRID:AB_940368) was used at a 1:20 dilution. For CD19 detection, APC anti-human CD19 antibody (Biolegend, 302212, RRID:AB_314242) was used at a 1:20 dilution. For branched N-glycan in human cancer cells, 2 μg/ml fluorescein-labeled PHA-L (Vectorlabs, FL-1111-2) was used. For bisecting GlcNAc in human cancer cells, 5 μg/ml fluorescein-labeled PHA-E (Vectorlabs, FL-1121-2) was used. For branched N-glycan in mouse cancer cells, 2 μg/ml biotinylated PHA-L (Vectorlabs, B-1115-2) was used, followed by streptavidin AF-647 (Thermo Fisher, S21374).

The canonical E-selectin ligands were analyzed at the tumor cell surface by flow cytometry using mAbs against sLeX (CSLEX1) or sLeA (121SLE) as described.⁵¹ (link) β-1,6-GlcNAc branches and poly-LacNAc were determined using biotinylated PHA-L and DSL, respectively (Vector Labs). As “isotype” controls, lectins were applied after sugar inhibition with bovine thyroglobulin (Sigma) and chitin hydrolysate (Vector Labs), respectively. Lectins were labeled with streptavidin-APC (Sigma) for flow cytometry.
To test their functional importance for endothelial adhesion and E-selectin binding, sialic-acid-containing sugar residues were enzymatically cleaved using 10 mU/mL ND (from Clostridium perfringens, Roche) added to the tumor cell culture at 37°C for 1h under serum-free conditions. Likewise, glycoproteins were cleaved by 1 mg/mL pronase (from Streptomyces griseus) added to the tumor cell culture at 37°C for 45 min under serum-free conditions. O-GalNAc-glycosylation was chemically inhibited by adding 2 mM GOB (Sigma) for 72 h to the culture medium (solvent control: cell culture medium). N-glycosylation was inhibited using 2 μM synthetic Sw (Sigma) for 72 h (solvent control: methanol). Detrimental effects of all treatments on tumor cell viability were excluded by propidium iodide uptake analyses (flow cytometry [FC]).