Macsquant x analyzer

Quantification of expression levels of CD19, TRAIL-R1, and TRAIL-R2 on the cell surface was analyzed by flow cytometry using QIFIKIT (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer´s protocol. Briefly, cells were washed with PBA (phosphate-buffered saline (PBS, Thermo Fisher Scientific, Waltham, MA, USA), 1% bovine serum albumin (BSA, Carl Roth, Karlsruhe, Germany), and 0.1% sodium-azide (Merck, Darmstadt, Germany). Then, 0.5 × 10⁶ cells were incubated with saturated concentrations of mouse antibodies against CD19 (#392502, Biolegend, San Diego, CA, USA), TRAIL-R1 (#307202, Biolegend, San Diego, CA, USA), and TRAIL-R2 (#307302, Biolegend, San Diego, CA, USA) for 1 h, respectively. After washing with PBA, cells were incubated for 30 min with a FITC-labelled anti-mouse antibody. Fluorescence was measured by flow cytometry using a MACSQuant X Analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany). Antigen density was assessed by generating a standard curve obtained by beads coated with defined amounts of mouse IgG. Data were analyzed by FlowJo software Version 10.7.1 (Becton, Dickinson and Company, Ashland, OR, USA).

Phenotyping was performed with antibodies presented in Supplementary Table 1. For macrophages staining experiment, cells were recovered from culture plates by gentle scrapping. TAM-R phenotyping was performed using the following Phycoerythrin (PE) conjugated antibodies: mouse IgG1 α-MerTK (Biolegend, clone 590H11G1E3), mouse IgG1 α-Tyro3 (R&D Systems, clone 96201), mouse IgG1 α-Axl (R&D Systems, clone 108724), or their respective isotypic controls. Cells were analyzed using MACSQuant Analyzer X (Miltenyi Biotech) and FlowJo software (BD). Gating was performed on single cells and live cells before applying the strategy depicted in Supplementary Figure 2A.

One hundred fifty thousand cells (SW-1710) and two hundred thousand cells (T-24) per well of a six well plate were seeded and reversely transfected with XtremeGENE™ HP (Roche). After 16 h cells were split into two wells. 48 h post transfection, supernatant and cells were collected, centrifuged at 1000 rpm for 5 min, washed with ice-cold 1x Annexin binding buffer (Serva, Heidelberg, Germany) and centrifuged again. The pellet was resuspended in 75 μl 1x Annexin binding buffer containing 4.5 μl Annexin V-APC (Serva) and 7.5 μl PI (1 mg/ml, Serva) and incubated for 15 min in the dark at RT. The suspension was diluted with 500 μl 1x Annexin binding buffer, centrifuged, washed and fixed with 0.5% methanol-free formaldehyde for 20 min on ice. The reaction was stopped with 500 μl 1x Annexin binding buffer. FACS measurements and analysis was performed using the MACSQuant Analyzer X and MACSQuantify Software (Miltenyi Biotec, Bergisch-Gladbach, Germany). In total, 50,000 cells/experiment were analyzed in three independent experiments.