Sc 69718

Single-cell suspensions of approximately 1×10⁶ elements were treated with 10% FcR blocking reagent (130-059-901, Miltenyi Biotec Inc. Auburn, CA) for 10 min at 4°C and then stained with anti-human nectin-1 (R1.302.12) (sc-69718, Santa Cruz Biotechnology, Inc. Dallas, TX), nectin-2 (R2.525) (sc-32804, Santa Cruz Biotechnology, Inc.) or HVEM (CW10) (sc-21718, Santa Cruz Biotechnology, Inc.). After applying the unconjugated primary Abs or their isotype controls, cells were stained with a secondary FITC-conjugated goat anti-mouse IgG (554001, BD Biosciences, San Jose, CA). For 3-O-Sulfated Heparan Sulfate (3-OS HS) analysis, cells were first fixed in 1% paraformaldehyde (PFA) for 10 min at 4°C followed by incubation with antibody HS4C3 (30 (link)) for 1 hour at 4°C. Next, cells were stained with unconjugated mouse anti-VSV (P5D4) (V5507, Sigma-Aldrich Corp. St. Louis, MO) for 30 min at 4°C. Finally, cells were stained with FITC goat anti-mouse Ig (554001, BD Biosciences) for 20 mins at 4°C. The analysis has been performed on the neuroblastoma panel for three independent times. Average mean fluorescence intensities (MFI) of nectin-1, nectin-2, HVEM and 3-OS HS relative to isotype (for netin-1, nectin-2 and HVEM) or unstained controls (for 3-OS HS) were calculated and presented as a heatmap in Figure 3c.