Anti mouse cd16 cd32

Bone marrow cells flushed from long bones were syringed five times through a 23-G needle and filtered through a 100μm nylon cell strainer. Red blood cells were lysed with 0.15 M NH₄Cl. Cells were resuspended in PBS and filtered through a 70μm strainer. Samples were incubated with Fixable Viability Dye eFluor780 (eBioscience, San Diego, CA) and Fc receptors were blocked with anti-mouse CD16/CD32 (Biolegend, San Diego, CA) at a dilution of 1:100. Cells were then incubated with anti-mouse CD115 APC, anti-mouse CD11b PERCP-Cy5.5, anti-mouse B220 Pe-Cy7, anti-mouse CD11c PE, and anti-mouse Gr-1 Brilliant Violet 421 (BioLegend) at a dilution of 1:100 in PBS/2% FBS. Samples were acquired on a FACSCanto II or LSR II SORP (BD Biosciences, San Jose, CA). Analysis was performed with FlowJo software (Tree Star, Ashland, OR).

Nine days post-injury, approximately 50 μL of blood was collected from the tail vein of the animals. Erythrocytes were depleted with ACK lysis solution. The cell pellet was then washed with FACS buffer (PBS, 10% BSA, 0.1% azide). 1x10⁶ cells were stained. The Fc portion was blocked using anti-mouse CD16/CD32 (Biolegend). Cell staining was performed by incubating a cocktail of antibodies for 30min at 4°C (Table 2). After washing, the cells were re-suspended in 200 μL FACS buffer. Precision counting beads (Biolegend) were added to the single-cell suspensions according to the manufacturer’s instructions to calculate the final cell concentrations. Cells were acquired using an LSRII Flow Cytometer (BD Pharminogen) and analyzed using Flow Jo software version 10.4. The gating strategy used can be found in the Supplementary Data (Supplementary Figure S5).

Peritoneal exudate cells (PECs) were obtained from the peritoneal cavity from distinct groups of mice 6 weeks p.i. of MLD-STZ. The cells were washed twice with physiological saline solution, and the red blood cells were lysed by resuspending the cells in Boyle's solution (0.17 M Tris and 0.16 M ammonium chloride). Following two washes, the viable cells were counted by trypan blue exclusion with a Neubauer hemocytometer. The PECs were adjusted (1 × 10⁶ cells), and Fc receptors were blocked with anti-mouse CD16/CD32 (Biolegend, CA, USA) and then stained with APC-F4/80, APC-CD11b, FITC-CD11c, FITC-MMR, PE-PDL-1, PE-PDL-2, PE-IL-4Rα, and PE-Gr1 (all from Biolegend, CA, USA) and incubated for 30 minutes at 4°C in FACSFlow staining buffer (Becton Dickinson). The cells were analyzed using a FACsCalibur and CellQuest Software (Becton Dickinson).

For surface cell staining, 5 × 10⁵ cells were incubated for 15–20 min with anti-mouse CD16/CD32 to block non-specific binding of immunoglobulins to Fc receptors expressed on monocytes, macrophages, and granulocytes (Biolegend, clone 93). Labeling was performed for 30 min with specific antibodies. The following antibodies were used: PE anti-mouse CD11b (Biolegend, clone M1/70), FITC anti-mouse F4/80 (eBioscience, clone BM8), and FITC anti-mouse Ly6G (Biolegend, clone 1A8). All staining was done on ice in PBS supplemented with 2% BSA followed by washing once in cold PBS. The FACS Vantage system (Becton-Dickinson, San Jose, CA, USA) was used to analyze samples.

In order to determine cell numbers in the bone marrow, around 100.000 bone marrow cells were freshly isolated. Aliquots of bone marrows were washed once in H/S (132 mM NaCl, 20 mM HEPES [pH 7.4], 5 mM KCl, 1 mM CaCl₂, 0.7 mM MgCl₂, 0.8 mM MgSO₄), resuspended in 100 μL H/S, Fc-receptors were blocked by incubation for 30 min with Fc-block (anti-mouse CD16/CD32, Biolegend #101302), samples were washed once and aliquots were stained with FITC-coupled anti-mouse CD45 antibodies (1:200, clone RA3-6B2; Biolegend, #103228) and APC-coupled antibodies against mouse CD4 (1:200, clone GK1.5; Biolegend, #17-0041-81) or APC-coupled antibodies against mouse CD8 (1:200, clone 53-6.7; Biolegend, #17-0081-81) or PE-coupled antibodies against mouse CD19 (1:200, clone GD5; Biolegend, #115508) or APC-coupled antibodies against mouse Ly6G (Gr1) (1:200, clone RB6-BL5; Biolegend, #103107). In addition, cells were stained with Alexa 647-coupled anti-mouse CD45 antibodies (1:200, clone 30-F11; Biolegend, #103228) and FITC-coupled antibodies against mouse Sca-1 (1:200, clone D7; Biolegend, #108105). Cells were stained for 1 h at 4°C, washed once in H/S and analyzed on a FACS-Calibur. We analyzed the expression of the above described markers on 100 000 cells by flow cytometry.

Colonic LP cells were incubated with anti-mouse CD16/CD32 (BioLegend) to block non-specific Fc receptors, followed by a cell surface staining with the corresponding mixture of fluorescently-labelled monoclonal antibodies. Seven-amino actinomycin D (BioLegend) was used to discriminate live and dead cells. The following antibodies conjugated with biotin, PE, peridinin chlorophyll-Cy5.5, PE-Cy7, APC, APC-Cy7, brilliant violet (BV) 421, BV510, BV650, BV711, or BV785 were used for flow cytometry: anti-B220, anti-CD4, anti-IA/IE, anti-Ly6G, anti-Siglec F (all from BD Biosciences), anti-CD3e, anti-CD8a, anti-CD11b, anti-CD11c, anti-CD45, anti-CD45.2, anti-CXCR4, anti-F4/80, anti-TER119 (all from BioLegend), anti-Ly6C, anti-NK-1.1, anti-Siglec F (all from Miltenyi Biotec, Auburn, CA) and anti-CCR2 (R&D Systems). Following extracellular staining, the cells were fixed and permeabilised with Cytofix/Cytoperm (BD Biosciences) and sequentially incubated with rabbit anti-collagen type I (Rockland, Limerick, PA), followed by Alexa Fluor 647-conjugated goat anti-rabbit IgG (Invitrogen). Data were acquired on LSRFortessa and processed using FlowJo software (Tree Star, Ashland, OR) with appropriate isotype controls to determine gating. Cell sorting was performed using FACSAria II (BD Biosciences).