U bottom 96 well plate

H1299 cells were detached with 2 mL of 1.5 mM EDTA in PBS (-/-), and 1 × 10⁵ cells were counted and transferred to a U-bottom 96 well plate (Sarstedt AG & Co. KG, Numbrecht, Germany). To quantify protein binding to cell surface receptors, cells were incubated with different concentrations of fluorescently labeled FS-Janus lectin and CBM40 solutions for 30 min at 4 °C and protected from light compared to PBS-treated cells as a negative control. For the saturation of fucose-binding sites, 32 nM FS-Janus lectin was pre-incubated with 25, 50, or 100 mM soluble l-fucose, for 30 min at RT, in the absence of light. At the end of pre-incubation, the solution was diluted 100 times and added to cells for 30 min at 4 °C, in the dark. Subsequently, cells were centrifuged at 1600× g for 3 min at 4 °C and washed twice with FACS buffer (PBS (-/-) supplemented with 3% FCS v/v). After the last washing step, the cells were re-suspended with FACS buffer and transferred to FACS tubes (Kisker Biotech GmbH Co. KG, Steinfurt, Germany) on ice and protected from light. The fluorescence intensity of treated cells was monitored at FACS Gallios (Beckman Coulter Inc., Brea, CA, USA) and further analyzed using FlowJo V.10.5.3.

H1299 cells were detached with 1.5 mM EDTA in PBS −/−, and 1 × 10⁵ cells were counted and transferred to a U-bottom 96-well plate (Sarstedt AG & Co.). To determine the binding of SaroL-1 to surface receptors, cells were incubated with fluorescently labelled protein for 30 min at 4 °C and protected from light, in comparison with PBS-treated cells as a negative control. To deplete cells of glucosylceramide-based glycosphingolipids, they were cultivated 72 h in the presence of 2 μM DL-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) (Sigma-Aldrich) to inhibit the synthesis of glucosylceramide-based GSLs^{87 (link)} and incubated with fluorescent SaroL-1. Subsequently, cells were centrifuged at 1600 × g for 3 min at 4 °C to remove unbound lectin. The samples were then washed two times with ice-cold FACS buffer (PBS (−/−) supplemented with 3% FCS (v/v)). After the last washing step, the cells were re-suspended with FACS buffer and transferred to FACS tubes (Kisker Biotech GmbH & Co) on ice and protected from light. The fluorescence intensity of treated cells was measured with FACS Gallios from Beckman Coulter. The samples were further analyzed using FlowJo V.10.5.3.

H1299 cells were detached with 2 mL of 1.5 mM EDTA in PBS, and 1 × 10⁵ cells were counted and transferred to a U-bottom 96 well plate (Sarstedt AG & Co. KG). To quantify protein binding to cell surface receptors, cells were incubated with different concentrations of fluorescently labeled RSL-MOA-Cy5 lectin for 30 min at 4 °C and protected from light compared to PBS-treated cells as a negative control. For the saturation of glycan-binding sites, 180 nM RSL-MOA-Cy5 was preincubated with 100 mM soluble l-fucose or with 100 mM 4-Nitrophenyl α-d-galactopyranoside (PNPG; Sigma-Aldrich, Chemie GmbH), for 30 min at RT and in the absence of light. At the end of pre-incubation, the solution was diluted 100 times and added to cells for 30 min at 4 °C, in the dark. Subsequently, cells were centrifuged at 1600 × g for 3 min at 4 °C and washed twice with FACS buffer (PBS supplemented with 3 % FCS v/v). After the last washing step, the cells were resuspended with FACS buffer and transferred to FACS tubes (Kisker Biotech GmbH Co. KG) on ice and protected from light. The fluorescence intensity of treated cells was monitored at FACS Gallios (Beckman Coulter Inc.) and further analyzed using FlowJo V.10.5.3.