Anti susd2 pe

Cells were single-cell dissociated using TrypLE Express and washed once in FACS buffer [PBS supplemented with 5% FBS]. The cells were then resuspended in 100 μL fresh FACS buffer, and incubated with antibodies for 30 min on ice. The following antibodies were used: anti-SUSD2-PE, 1:100 (BioLegend, 327406); anti-CD75-eFluor 660, 1:100 (Thermo Fisher, 50-0759-42); anti-ITG6-FITC, 1:100 (Miltenyi, 130-097-245); anti-EGFR-APC, 1:20 (BioLegend, 352905); anti-HLA-G-PE, 1:100 (Abcam, ab24384). Following antibody incubation, the cells were washed once with FACS buffer, resuspended in fresh FACS buffer, and passed through a cell strainer. Unstained cells that have undergone the same procedures were used as controls. Flow cytometry was performed using a BD LSRFortessa X-20, and the data were analyzed using the FlowJo software.

Cells were single cell dissociated using TrypLE Express and washed once in FACS buffer [PBS supplemented with 5% FBS]. The cells were then resuspended in 100 μL fresh FACS buffer, and incubated with antibodies for 30 min on ice. The following naïve-specific cell-surface antibodies were used: anti-SUSD2-PE, 1:100 (BioLegend, 327406) and anti-CD75-eFluor 660, 1:100 (Thermo Fisher, 50-0759-42). Following antibody incubation, the cells were washed once with FACS buffer, resuspended in fresh FACS buffer, and passed through a cell strainer. Cell debris was excluded by FSC vs. SSC gates and single cells were gated by FSC-A vs. FSC-W. Naïve cell-surface markers were analyzed with anti-CD75-eFluor 660 (APC channel) and anti-SUSD2-PE. Primed cells and unstained cells that have undergone the same procedures were used as controls. Flow cytometry analysis was performed using a BD LSRFortessa X-20 and the data were analyzed using the FlowJo software.

Cells were single-cell dissociated using TrypLE Express and washed once in FACS buffer [PBS supplemented with 5% FBS]. The cells were then resuspended in 100 μL fresh FACS buffer, and incubated with antibodies for 30 min on ice. The following antibodies were used: anti-SUSD2-PE, 1:100 (BioLegend, 327406); anti-CD75-eFluor 660, 1:100 (Thermo Fisher, 50-0759-42); anti-ITGA6-FITC, 1:100 (Miltenyi, 130-097-245); anti-EGFR-APC, 1:20 (BioLegend, 352905); anti-Annexin V-FITC, 1:10 (Thermo Fisher, BMS147FI). Following antibody incubation, the cells were washed once with FACS buffer, resuspended in fresh FACS buffer, and passed through a cell strainer. Unstained cells that have undergone the same procedures were used as controls. Cell cycle analysis was performed using the Vybrant DyeCycle Violet Ready Flow Reagent (Thermo Fisher, R37172) according to the manufacturer’s instructions. 100 µM Verapamil (Sigma-Aldrich, V4629) was added during incubation to prevent dye efflux. Flow cytometry was performed using a BD LSRFortessa X-20 and the data were analyzed using the FACSDiva v9.0 or FlowJo v10.8.1 software.