Apc cy7 rat igg2a

Single cell dissociation was performed through enzymatic digestion with 600 U ml^-1 collagenase (Sigma) and 200 U ml^-1 hyaluronidase (Sigma) for 90 min at 37 °C. Cells were further dissociated in TrypLE (Gibco) for 3 min, in 5 mg ml^-1 dispase (Roche) and 0.1 mg ml^-1 DNase I (Sigma) for 5 min, and then in 0.63% NH₄Cl and filtered through a 40 μm cell strainer to obtain a single cell preparation for FACS. Cell labelling and flow cytometry were performed as described previously (Koren et al., 2015 (link)) using LSRII or FACS ARIA flow cytometers (BD). Dead cells (DAPI⁺), and CD45⁺/CD31⁺/Ter119⁺ (Lin⁺) non-epithelial cells were excluded. The following antibodies were all purchased from Biolegend and were used at a 1:100 final concentration: PE/Cy7 anti-mouse CD24, PE/Cy7 anti-mouse Epcam, AlexaFluor700 anti-mouse/rat CD29, APC/Cy7 anti-mouse CD49f, lineage markers: APC anti-mouse CD31, APC anti-mouse Ter119, APC anti-mouse CD45, isotype controls: PE rat IgM, PerCP/Cy5.5 rat IgGa, PE/Cy7 rat IgG2a, APC/Cy7 rat IgG2a, APC rat IgG2b. The purity of sorted populations was approximately 95%. The results were analyzed using FlowJo software (v10, BD).

The cells obtained from the wounded tissues were incubated with Anti-Mouse CD16/CD32 (clone 2.4G2, BD Biosciences, Franklin Lakes, NJ, USA) on ice for 15 min in PBS that contained 1% FCS and 0.1% sodium azide. The cells were stained with Pacific blue-anti-CD45 monoclonal antibody (mAb) (clone 30-F11, BioLegend, San Diego, CA, USA), APC-anti-CD11b mAb (clone M1/70, BioLegend, San Diego, CA, USA), APC/Cy7-anti-Ly6G mAb (clone 1A8, BioLegend, San Diego, CA, USA), and FITC-anti-F4/80 mAb (clone BM8, BioLegend, San Diego, CA, USA). Isotype-matched irrelevant IgG (Pacific: blue Rat IgG2b, κ Isotype Ctrl mAb, APC Rat IgG2b, κ Isotype Ctrl, APC/Cy7 Rat IgG2a, κ Isotype Ctrl, and FITC Rat IgG2a, κ Isotype Ctrl, BioLegend, San Diego, CA, USA) was used for control staining. The stained cells were analyzed using a BD FACS CantoTM II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed using FACS Diva software. Neutrophils and macrophages were identified as CD45⁺CD11b⁺Ly6G⁺ cells and CD45⁺CD11b⁺F4/80⁺ cells, respectively. The number of neutrophils and macrophages was estimated by multiplying the total leukocyte number by the proportion of each fraction.