Lsrii flow cytometer

Serum levels of various cytokines (IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, IL-21, IL-22, IFN-γ, and tumor necrosis factor (TNF)-α) were determined using the LEGENDplex™ Mouse Th Cytokine Panel (13-plex) array (Biolegend) according to the manufacturer’s protocol. The data were collected on a LSR II flow cytometer and analyzed using the LEGENDplex™ software version 7.0 (Biolegend).

Total tumor cells were resuspended in PBS and centrifuged at 500 × g for 5 minutes. The samples were resuspended in 100 μl LIVE/DEAD Fixable Blue Dead Cell Stain kit (Invitrogen) and incubated for 15 minutes at RT in the dark, and 200 μl of PBS was added to the tube and centrifuged at 500 × g for 5 minutes. The pellet was resuspended in PBS and centrifuged at 500 × g for 5 minutes. Cells were stained with following antibodies at 1:25 dilution: F4/80 Alexa Fluor 488 (Bio-rad); 1:100 dilutions: CD3 APC (Tonbo Bioscience), MR PE, Ly-6C Alexa Fluor 700, NK1.1 APC-Cy7, Ly-6G Pacific Blue, CD4 PerCP/Cy5.5, CD8 APC-Cy7, CD279 PE (Biolegend); 1:200 dilution: CD274 PE-Cy7 (Biolegend) in FACS buffer (5% FBS in PBS) for 15 minutes at 4 °C in dark. 100 μl FACS buffer was added into the tubes and centrifuged at 500 × g for 5 minutes. Cells were washed with 150 μl FACS buffer and centrifuged at 500 × g for 5 minutes twice. The pellet was resuspended in 200 μl FACS buffer and run with LSRII flow cytometer (Biolegend). Results were analyzed with FlowJo (Treestar).

Single-cell suspensions were prepared, counted, and analyzed using specific antibodies (S2 Table) by an LSRII flow cytometer (BD Biosciences), as previously described [91 (link)]. Data were analyzed using the FlowJo software (Tree Star). Apoptotic cells were detected using the PE Annexin V Apoptosis Detection Kit with 7-AAD according to the manufacturer’s protocol (BioLegend) and analyzed using an LSRII flow cytometer.