Anti mouse cd16 cd32 mab

Splenocytes, lymph node cells, and tumor cells isolated from mice were incubated with anti-mouse CD16/CD32 mAb (15 min, 4°C, eBioscience). Splenocytes were stained with the LIVE/DEAD FixableViolet Dead Staining Kit (Thermo Fisher Scientific, Inc.). Subsequently, the cells were divided and stained with cocktails of fluorochrome-conjugated monoclonal antibodies: anti-CD3 PE-CF594, CD19 PE-CF594, and CD49b PE-CF594 (all from BD Biosciences); CD45 V500, CD11b PerCP-Cy5.5, CD11c BV650, F4/80 Alexa Fluor 700, CD86 PE-Cy7, and MHC II APC-Cy7 (all from BioLegend) for myeloid cell identification; and CD45 V500, CD4 PerCp-Cy5.5, CD8 PE-Cy7, CD49b PE-CF594, CD44 PE, and CD62L BV605 for lymphocyte identification. Cells were incubated with antibodies for 45 min at 4°C. Additionally, the percentage of Treg cells was determined among the splenocytes. For this purpose, the cells were fixed using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) and then incubated with anti-FoxP3 APC antibodies (eBioscience) [28 (link)]. To identify dead cells in lymph node cells and tumor cells, 50 μl of DAPI dye solution (1 μg/ml, Molecular Probes) was added to the samples immediately before analysis. The analysis was performed using a BD LSRFortessa Cell Analyzer (Becton Dickinson, Cat. No. 649225B5) with the BD FACSDiva software 8.0 and the NovoExpress software 1.3.0 (ACEA Biosciences, Inc.).

NK cells were aseptically isolated by mechanical dispersion of the whole GD8 BALB/c spleen in RPMI-1640 medium supplemented with 1% FBS and penicillin/streptomycin (100 U/ml). Cell suspensions were subsequently passed through a 37 μm nylon mesh followed by density gradient separation using HISTOPAQUE 1083 (Sigma-Aldrich), according to the manufacturer's instructions. Briefly, cell suspensions were carefully loaded onto the HISTOPAQUE 1083 surface and centrifuged at 2000 r.p.m. for exactly 30 min at room temperature. After centrifugation, the opaque interface containing the mononuclear cells was carefully aspirated, washed with RPMI-1640 medium and resuspended in PBS containing 0.2% BSA. Cells were then pretreated with anti-mouse CD16/CD32 mAb (eBioscience) for 10 min on ice and incubated with APC-conjugated anti-CD49b (BD Biosciences, San Jose, CA, USA) and PE-conjugated anti-CD3 (eBioscience) mAbs for 30 min at 4 °C. After incubation, cells were washed once with PBS containing 0.2% BSA and sorted by a FACSAria instrument (BD Biosciences, Franklin Lakes, NJ, USA). Postsort NK cell purity was routinely more than 95% (data not shown).