Cd45 microbeads

We performed SA-βGal staining using kit (Cell Signaling; cat. no. 9860). Briefly, skeletal muscle primary cells were isolated from 3-months-old WT and DMD rats as described above. To exclude the leukocytes including macrophages, we performed MACS separation using CD45 microbeads (Miltenyi Biotec; Bergisch Gladbach, Germany). After elimination of leukocytes, cells were fixed for 10 min with fixation buffer, followed by 15 h incubation in staining solution at 37 °C.

Lizard (L. lugubris) tail and limb tissues were washed three times in 10% povidone-iodine solution and washed once in HBSS supplemented with 100 units/mL penicillin, 100 µg/mL streptomycin, and 250 ng/mL fungizone antimycotic. Washed tissues were incubated in 0.1% EDTA in HBSS for 45 min at room temperature with agitation, and scales/epidermis were peeled from each tissue piece with forceps and discarded. Prepared tissues were then washed extensively in HBSS, minced, and digested in 1 mg/mL trypsin and 1 mg/mL collagenase II for 1 h at 37 °C. Immune, muscle-related, and endothelial cells were depleted by passing cell suspensions through the following MACS® (Miltenyi Biotec) magnetic beads: CD144 (VE-Cadherin) MicroBeads (PN: 130-097-857), Anti-Integrin α−7 MicroBeads (PN: 130-104-26), CD45 MicroBeads (PN: 130-052-301), CD326 (EpCAM) MicroBeads (PN: 130-105-958), according to the manufacturer’s instructions. Cell/bead suspensions were loaded onto LD columns (Miltenyi Biotec, PN: 130-042-901) and placed on MidiMACS™ Separator magnets (Miltenyi Biotec, PN: 130-042-301). Enriched fibroblast suspensions were collected in lizard cell culture medium (Dulbecco’s Modified Eagle Media (DMEM)/Ham’s F12, 2 mM Glutamax, 0.1 µM dexamethasone, 40 µg/mL proline, 50 µg/mL ascorbate, and 10 µg/mL ITS+ supplement).

Thymus of WT and Kcnk18^G339R mice was homogenized by enzymatic digestion. Therefore, the thymus was dissected into small pieces by cutting with scissors in 5 mL RPMI containing 2% FCS. 0.5 mg/mL collagenase D and 20 µg/mL DNase I were added to the tissue and incubated for 45 min at 37 °C on an orbital shaker. Digestion was stopped by 5 mL of 10 mM EDTA, followed by incubation for 5 min at RT. Thymus homogenate was then rinsed through a 70 µm cell strainer and washed with 20 mL RPMI + 2% FCS. To isolate CD11c⁺ cells, CD11c MicroBeads (MACS, Miltenyi Biotec) were used in a MACS separation according to the manufacturer’s protocol. After isolation, cells were stained for flow cytometry with antibodies directed to CD8, CD11b, CD11c, CD45R/B220, CD86, MHC-II, SIRPα. For CD45^– mTEC isolation, we used the CD11c^– flow-through from CD11c MACS isolation in a negative selection using CD45 MicroBeads (MACS, Miltenyi Biotec) according to the manufacturer’s protocol. After MACS isolation, CD45^– mTEC was used for flow cytometry analysis (CD45, CD80, BP-1, EpCAM, MHC-II).

Fibroblasts were isolated from RA-ILD or control lung as previously described^{34 (link),35 (link)}. Briefly, lung tissue was minced and digested in collagenase buffer. Cell pellets were resuspended and cultured for 7–10 days. Cells were CD45-depleted using CD45 microbeads (Miltenyi Biotec) to remove hematopoietic cell populations. Fibroblasts were cultured in Dulbecco’s Modified Eagle Medium (Corning) containing 10% fetal bovine serum (Corning), 100 IU of penicillin and 100 μg/ml streptomycin (Corning), 292 μg/ml L-glutamine (Corning), and 100 μg/ml Primocin (InVivoGen) in humidified incubators at 37 °C and 10% CO₂. Cells were periodically tested for Mycoplasma spp. contamination using a commercially available kit (ThermoFisher Scientific).