4 6 diamidino 2 phenylindole dilactate dapi

Peripheral blood was drawn into 3.8% sodium citrate anticoagulant monovettes and separated by density gradient centrifugation (Biocoll density 1.077 g/mL, Biochrome). The mononuclear cell fraction was discarded, and neutrophils were isolated by sedimentation over 1% polyvinyl alcohol, followed by hypotonic (0.2% NaCl) lysis of erythrocytes. In view of the emerging diversity of circulating neutrophil subtypes in humans, it should be noted that high-density neutrophils were investigated in this study.
The purity of the isolated neutrophils (>95%) was estimated with flow cytometry after staining with anti-CD66b (Beckman Coulter, Krefeld, Germany). Viability Dye eFluor™ 780 (eBioscience, Affymetrix, Santa Clara, CA, USA) or 4′,6-Diamidino-2-Phenylindole, Dilactate (DAPI) (BioLegend, San Diego, CA, USA) were used to determine viable cells. (Supplementary Figure S1A). Data were collected and analyzed with the BD FACS Canto system and BD FACS Diva 6.0 software (BD Biosciences, BD, Franklin Lakes, NJ, USA).

To prepare single cell suspension, muscles were dissected from mice and finely minced with a scalpel in a dish containing DMEM, as previously described [12 (link)]. Next, minced muscles were digested in a solution of Collagenase type IV (Worthington, Lakewood, NJ, USA) 1 mg/mLin DMEM, (10 mL/g of muscle) for 1h 30 min in shaking water bath at 37 °C. Then, digested muscles were passed through a 70 µm cell strainer first and then through a 40 µm cell strainer, to exclude cell debris. Muscle single cell suspension was then resuspended in FACS buffer (PBS 1% FBS) and incubated 30 min on ice with the following antibodies: anti-CD45 (Biolegend, clone 30-F11, 1:6000), anti-Ly6G (eBioscience, clone 1A8 and clone RB6-8C5, 1:3000), anti-Ly6c (Biolegend, clone HK1.4, 1:100), anti-F4/80 (Biolegend, clone BM8, 1:1000), anti-CD206 (MMR) (Biolegend, clone C068C2, 1:50), anti-CD11b (Biolegend, clone M1/70, 1:3000). Cell viability was assessed with 4′,6-diamidino-2-phenylindole dilactate (DAPI) (BioLegend).
For CFSE analysis isolated SCs were stained with CFSE (ThermoFisher Scientific, Waltham, MA, USA) 5 µm for 20′ at 37 °C in dark prior to culture. Samples were processed using a Dako CyAn ADP flow cytometer and acquired data were analyzed using FlowJo software version 10 (FlowJo LLC, Ashland, OR, USA).

Muscle single-cell suspensions were obtained as previously described [13 (link), 24 (link)]. Briefly, mice were killed by cervical dislocation and perfused with PBS, and the hind limb musculature (tibialis anterior and gastrocnemius) was excised, weighed, cut into 1-mm pieces and digested with 1 mg/ml Collagenase type 4 (Worthington) for 1.5 h in a pre-warmed (37 °C) water bath with agitation. Muscles from each mouse were processed individually. The digested muscle preparation was first passed through a 70 μm cell strainer (BD Falcon) and then through a 40 μm cell strainer, washed in Dulbecco’s modified eagle’s medium (DMEM)/10% fetal bovine serum (FBS) and resuspended in FACS buffer (PBS 1%FBS). Single cells were counted with a hemocytometer and incubated with anti-CD16/32 (clone 93, Biolegend) to block non-specific binding to Fc receptors. Samples were then stained with the relevant antibodies anti-CD45 (clone 30-F11); anti-Ly6C (clone HK14); anti-Ly6g (clone 1A8); anti-F4/80 (clone BM8) all from Biolegend). Cell viability was assessed with 4′,6-diamidino-2-phenylindole, dilactate (DAPI) (Biolegend). Samples were acquired with a CyAn ADP (DAKO), and acquired data were analyzed using FlowJo software version 10.