Liberase ci

Spleen and LNs of DO11.10 mice were harvested, digested in RPMI-1640 medium (GIBCO, Paisley, UK) containing liberase CI (0.42 mg/ml (1.67 U/ml), Roche) and DNase 1 (0.2 mg/ml (400 U/ml), Roche) and passed through a 40 µm cell strainer (BD Biosciences). Single-cell suspensions were incubated for 20 min with anti-CD4⁺-coupled magnetic beads and isolated using a MACS column and separator (all from Miltenyi Biotech, Gladbach, Germany). Isolated CD4⁺ T cells (purity > 90%) were stained with 2 µM CFSE (Invitrogen) for 12 min at 37°. Subsequently, T cells were co-cultured for 3 days with FACS-sorted DCs, at a T cell:DC ratio of 5∶1, in presence of 100 µM OVA peptide (residues 323-339, GenScript, Piscataway, NJ, USA). Co-cultures were performed in RPMI-1640 medium (GIBCO) supplemented with 10% fetal bovine serum (FBS; GIBCO), 50 µM β-mercaptoethanol (Sigma), 15 mM HEPES (GIBCO), 1 mM Na-pyruvate (GIBCO), antibiotic/antimycotic solution (Fluka, Buchs, Switzerland) and 2 mM L-glutamine (Fluka).

Single-cell suspensions of hind-limb muscles containing the femoral arteries, and paw tissues were generated by careful mincing tissues and subsequent digestion at 37°C for 1 hour in DMEM (Life Technologies) in the presence of 250 µg/ml liberase CI (Roche) plus 50 µg/ml DNase I (Roche). After filtration through a 40 µm nylon cell strainer (BD Biosciences) and centrifugation for 5 min at 400 g, the cells were washed and treated with ammonium-chloride-based erythrocyte lysis buffer. All flow cytometric measurements were performed in the buffer containing PBS with 2 mM EDTA and 2 % FBS (Life Technologies) on a Becton Dickinson LSRFortessa flow cytometer and were analyzed by FlowJo software (Treestar Inc.). Csf1r-EGFP positive and MF800 positive signals have been detected through FITC/Alexa Fluor488 (Excitation: 488 nm laser with 50 mW power; Emission: 530 ± 15 nm filter) and APC-Cy7/APC-H7 (Excitation: 640 nm laser with 40 mW power; Emission: 780 ± 30 nm filter) channels, respectively.

Lungs were isolated and digested in an enzyme mixture of 6 mL of RPMI 1640 containing Liberase CI (0.5 mg/mL) (Roche Diagnostics, Indianapolis, IN, USA) and DNase I (0.5 mg/mL) (Sigma-Aldrich, St. Louis, MO, USA) for 45 min at 37 °C. The digested lung tissue was then homogenized with a 3-mL syringe plunger through a 70-μm cell strainer, and red blood cells were lysed with ACK lysis buffer (prepared in house).

Spleens were injected with Liberase CI (Roche, Indianapolis, IN, USA) and incubated at 37C for 30 minutes, after which they were meshed through a 70-micron strainer and washed. The pellets were resuspended in 30% BSA in PBS, overlayed with PBS/0.1% BSA, and spun for 15 minutes at 9,500g at 4C. The low-density cells at the interface were collected, washed, and used for further analysis.