Bead based assay

Multiplex cytokine analysis was performed using a bead-based assay (BioLegend, Koblenz, Germany) according to the vendor’s instructions. Briefly, lavage supernatants were incubated with antibody-coated beads, and mean fluorescence intensities (MFI) of each bead population (representing a single analyte) were determined using flow cytometry (CytoFLEX S; Beckman-Coulter, Krefeld, Germany). Total cytokine concentrations in picogram per milliliter were calculated against a known standard and using 5-log fitting with dedicated LEGENpPlex analysis software (version 8; BioLegend, Amsterdam, Netherlands). To optimize compact data display in a single heatmap, data were normalized against the lavage of mice receiving untreated saline as control.

Multiplex chemokine, cytokine, and growth factor quantification was performed using a bead-based assay (BioLegend, Amsterdam, The Netherlands) according to the vendor’s instructions. Briefly, supernatants of the in vitro co-culture assays and ex vivo GBM tissue cultures were incubated with beads, and mean fluorescence intensities (MFI) of each bead population (representing a single analyte) were determined using flow cytometry (CytoFLEX S; Beckman Coulter, Krefeld, Germany). Total analyte concentrations were calculated in picogram per milliliter against a known standard using 5-log fitting with dedicated VigeneTech software. Statistical analysis was performed by parametric paired t-tests for each analyte and donor.