Anti cd11b bv421

Double staining of annexin V and PI was performed using Annexin V Apoptosis detection set phycoerythrin (PE)-Cy7 (eBioscience, San Diego, CA, USA) or Brilliant Violet 421™ (BV421) Annexin V (BioLegend, San Diego, CA, USA), and activated caspases detection was performed using a FLICA 660 caspase-1 or caspase-3/-7 assay kit (Immunochemistry Technologies, Bloomington, MN, USA) according to the manufacturer's protocol. Stained cells were analyzed with a flow cytometer Cytomics FC 500 (Beckman Coulter, Fullerton, CA, USA). Triple staining of CD11b, Ly6C, and Ly6G for 32D/TetOff-p210 cells was performed using anti-CD11b-BV421, anti-Ly6C-allophycocyanin (APC) (BD Biosciences), and anti-Ly6G-PE (BD Biosciences), respectively. Stained cells were analyzed with a high-speed cell sorter MoFlo Astrios^EQ (Beckman Coulter, Fullerton, CA, USA). Triple staining of CD11b, Ly6C, and Ly6G for spleen cells from mice was performed using anti-CD11b-PE (BD Biosciences) anti-Ly6C-APC (BD Biosciences), and anti-Ly6G-fluorescein isothiocyanate (FITC) (BD Biosciences), respectively. Stained cells were analyzed with a flow cytometer Cytomics FC 500 (Beckman Coulter).

Tumor‐bearing mice were anaesthetized by IP injection of 100 μl sodium pentobarbital. Both femoral arteries were opened, and the animals were perfused through the left cardiac ventricle with 50 ml cold PBS. Tumors were harvested and incubated with RPMI medium containing 10% FCS, 2.5 mg/ml collagenase D, and 5 U/ml DNase I for 30 min at 37°C. After incubation, the digested tissue was passed through a cell strainer and thoroughly washed. Cells were stained with the following monoclonal antibodies: anti‐CD45 Alexa Fluor^® 700 (eBioscience), anti‐CD11b BV421 (BD), anti‐Ly6C Alexa Fluor^® 647 (AbD Serotec), anti‐Ly6G FITC (BD), anti‐MHCII PerCP‐Cy5.5 (Biolegend), and anti‐MRC1 PE (Biolegend). For the MRC1 staining, cells were fixed and permeabilized by using the LEUCOPERM™ reagents (AbD Serotec). As a control, cells were stained with the appropriate isotype control. To exclude dead cells, cells were stained with the Zombie Yellow™ dye (Biolegend). Data acquisition was performed on the BD LSRFortessa and analysis was performed with FlowJo_V10.

Tumor cell suspensions (4 × 10⁶) prepared by mechanical and enzymatic dissociation^{16 (link)} were stained in 96-wells round bottom plates with live/dead staining (Blue fluorescent reactive dye, #L34962 Invitrogen) during 20 min at room temperature. For flow-cytometry analysis and sorting, Fc receptors were blocked with anti-FcR (anti-CD16 and CD32, at 5 µg/ml, Biolegend #101339). After two washes in PBS 2% FCS, cells were stained with the following antibodies (all used at 1:100): anti-CD11b-BV421 (#562605), CD64-APC (#558539), CD11c-PeCy7 (#557401), TCRβ-BV605 (#562840) and CD4-BV711 (#563050) all purchased from BD Pharmingen; anti-CD45-AF700 (#103128), Ly6C-APCCy7 (#128025), Ly6G-BV510 (#127633), F4/80 BV650 (#123149), CD206-PE (#141706), IA/IE-BV785 (#107645) all purchased from Biolegend, and CD8-PerCPef710 (#46-0081-82) purchased from eBioscience. After washing, cells were fixed in 1% PFA, stored at 4 °C, and acquired the next day on LSR II or FORTESSA (BD Bioscience). For detection of phosphorylated proteins, cell suspensions were stimulated 3 h with DMXAA 250 µg/ml, fixed immediately in PFA 4%, permeabilized with frozen methanol 90%, stained overnight with 1:100 pTBK1-PE (#13498) antibodies (purchased from Cell signaling), then washed and further stained for multicolor flow cytometry.

The isolated immunocytes were blocked with purified rat anti-mouse CD16/32 antibodies (BD Biosciences, USA) for 30 min at 4°C, then stained with antibodies for 30 min at 4°C, washed and analyzed by a BD FACSVerse flow cytometer (BD Biosciences, USA). The following monoclonal anti-mouse antibodies were used: anti-CD11b-BV421, anti-LY6C-APC, anti-LY6G-PE, anti-CD3-PerCP, anti-CD4-APC-H7, and anti-CD8-FITC (BD Biosciences, USA). The labeling cells were assessed using a BD FACSVerse flow cytometer (BD Biosciences, USA). The acquired data were analyzed using FlowJo 7.6 software (TreeStar, Inc.).

Fresh tumor tissue was minced with scissors and mechanically dissociated in a non-enzymatic solution using the GentleMACS dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany) as previously described [41 (link)]. To precisely identify the cell types expressing active caspase-1, 5 × 10⁵ cells were incubated for 30 min at 37 °C with the fluorescent probe FAM-YVAD-FMK (1:150) in Dulbecco’s modified Eagle Medium (DMEM)/10% fetal Bovine serum (FBS), and washed twice with Phosphate Buffered Saline (PBS)/0.1% Bovine Serum Albumin (BSA). Then the cells were incubated for 30 min at 4 °C in the presence of the viability dye FVS eFluor^TM 780 (BD Biosciences), and the following antibodies: Epcam-APC (Biolegend), anti-CD11b-BV421 (BD Biosciences), or the isotype control antibodies. To detect IL-18 receptors on TILs, 1 × 10⁶ cells were incubated for 30 min at 4 °C with FVS 780 (BD Biosciences) and the following antibodies: IL-18α PE-Vio 770 (Miltenyi Biotec), CD3-BUV395 (BD Biosciences), CD4-BUV496 (BD Biosciences), CD8-APC (Biolegend), or the isotype control antibodies. After washing twice with PBS/0.1% BSA, positive cells were acquired in the viable cell gate on a LSR Fortessa × 20 flow cytometer (BD Biosciences) and analyzed using BD FACS Diva software 8.0.2 version.