Cd64 apc

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.

Fascia of the TA muscles was removed. Muscles were dissociated and digested in RPMI medium containing 0.2% of collagenase B (Roche Diagnostics GmbH 11088815001) at 37 °C for 1 h and passed through a 70 μm and a 30 μm cell strainer. CD45⁺ cells were isolated using magnetic beads (Miltenyi Biotec 130-052-301) and incubated with FcR blocking reagent (Miltenyi Biotec 130-059-901) for 20 min at 4 °C in PBS 2% FBS. Cells were then stained with Ly6C-PE (eBioscience 12-5932) and CD64-APC (BD Pharmingen 558539) antibodies for 30 min at 4 °C. MPs were analyzed or sorted using a FACS Aria III cell sorter (BD Biosciences) (gating strategy is shown Figure S1). In some experiments, Ly6C⁺ and Ly6C⁻ MPs were cytospined on starfrost (Knitterglaser, Bielefeld, Germany) slides and immunostained.

Mpcs were labelled using the CellVue Claret Far Red kit (Sigma-Alrich MinClaret) by following the manufacturer’s instructions (Sigma-Aldrich) and treated with staurosporin at 5 μM for 4 h, in order to induce apoptosis. M1 and M2c polarized MPs were incubated with apoptotic mpcs at a 1:3 ratio for 30 min at 4 °C or 6 h or 16 h at 37 °C. After three PBS washings, MPs were detached using trypsin and a cell scraper and cells were labelled with a CD64-APC (BD Pharmingen 558539) and analyzed by flow cytometry using a FACS Aria III cell sorter (BD Biosciences). The double-positive cells (CD64⁺/Far Red⁺ cells) were phagocytic MPs, whereas the CD64⁺/Far Red⁻ cells were nonphagocytic MPs. To exclude MPs that have bound, but not ingested, apoptotic cells, we subtracted the percentage of double-positive cells observed at 4 °C from the value observed at 37 °C. In some experiments, MPs were treated with 1 μg/mL of cytochalasin D (Sigma-Aldrich C8273), 45 min before adding apoptotic mpcs, and with the added mpcs.

Peripheral blood, spleens, and mesenteric lymph nodes (mLN) were collected 24 h after CLP surgery or sham surgery to obtain single cells for flow cytometry. Cells were stained for 30 min at 4°C with the following antibodies: CD45-Percp, CD3-FITC, CD4-Percp, CD8-APC, B220-PE, CD11c-APC, CD11b-FITC, F4/80-PE, and Ly6G-PE. Following incubation, red blood cells were lysed and washed 3 times with a FACS buffer before collecting data. Immunophenotyping analysis of the BMDMs was conducted using a flow cytometry technique. Briefly, 100 μl of cell suspension was incubated for 5 min with 5 μl of Fc blocker (Innovex, USA). Then, 1 μl of monoclonal antibodies (CD11b-FITC, F4/80-PE, F4/80-FITC, MHCII-FITC, CD80-PE, CD80-APC, CD16/32-Percp-cy5.5, and CD64-APC, BD, USA) were added and incubated at 4°C for 30 min and washed 3 times with the FACS buffer. All samples were analyzed using a FACSCalibur flow cytometer (BD, USA).

Bone marrow samples were obtained by standard bone marrow puncture using sterile heparin anticoagulant tubes. Bone marrow samples were filtered using flow cytometry tubes. CD14-FITC (Cat No.: 555397), CD68-PE (Cat No.: 565595), CD64-APC (Cat No.: 561189), CD40-PEcy7 (Cat No.: 561215), CD206-PE (Cat No.: 555954), CD163-PEcy7 (Cat No.: 556018), and isotype control antibodies (BD Biosciences, USA) were added to the tubes. The samples were then stained for 15 min in the dark at room temperature. After red blood cell lysis, the cells were washed with PBS. Finally, the cells were detected using a FACSCalibur flow cytometer (BD Biosciences, USA). Data analysis was performed using the Cell Quest software (Becton Dickinson, version 3.1).
Macrophages were defined as CD14⁺CD68⁺ cells. M1 macrophages were defined as CD64⁺CD40⁺ macrophages. M2 macrophages were defined as CD206⁺CD163⁺ macrophages (detail in Supplemental Figure 1).