233 fb cf

Satellite cell isolation and differentiation were performed as described previously with minor modifications (Hindi et al., 2017 (link)). Briefly, gastrocnemius and quadriceps muscles were excised from Lrrc8a^flfl mice (8–10 weeks old) and washed twice with 1XPBS supplemented with 1% penicillin-streptomycin and fungizone (300 µl/100 ml). Muscle tissue was incubated in DMEM-F12 media supplemented with collagenase II (2 mg/ml), 1% penicillin-streptomycin and fungizone (300 ul/100 ml) and incubated at shaker for 90 min at 37°C. Tissue was washed with 1X PBS and incubated again with DMEM-F12 media supplemented with collagenase II (1 mg/ml), dispase (0.5 mg/ml), 1% penicillin-streptomycin and fungizone (300 µl/100 ml) in a shaker for 30 min at 37°C. Subsequently, the tissue was minced and passed through a cell strainer (70 µm), and after centrifugation; satellite cells were plated on BD Matrigel‐coated dishes. Cells were stimulated to differentiate into myoblasts in DMEM‐F12, 20% fetal bovine serum (FBS), 40 ng/ml basic fibroblast growth factor (bfgf, R and D Systems, 233‐FB/CF), 1X non‐essential amino acids, 0.14 mM β‐mercaptoethanol, 1X penicillin/streptomycin, and Fungizone. Myoblasts were maintained with 10 ng/ml bfgf and then differentiated in DMEM‐F12, 2% FBS, 1X insulin–transferrin–selenium, when 80% confluency was reached.

Satellite cell isolation and differentiation were performed as described previously with minor modifications [10 (link)]. Briefly, the skeletal muscles of gastrocnemius and quadriceps were excised from C57BL/6J wildtype at 8–10 weeks of age. The muscles were washed twice with 1× PBS supplemented with 1% penicillin-streptomycin and fungizone (300 μL/100 mL). DMEM-F12 media with collagenase II (2 mg/mL), 1% penicillin-streptomycin, and fungizone (300 uL/100 mL) was added to the muscles, and the muscles were shaken for 90 min at 37 °C. The tissue washing process was repeated, using DMEM-F12 media, same specifications as before except collagenase II was changed to 0.5 mg/mL, in a shaker for 30 min at 37 °C. Following this, the tissue was cut and passed through a cell strainer (70 μm). Following centrifugation, satellite cells were plated on BD Matrigel-coated dishes. Cells were differentiated into myoblasts through the usage of a mixture of DMEM-F12, 20% fetal bovine serum (FBS), 40 ng/mL basic fibroblast growth factor (bfgf, R and D Systems, 233-FB/CF), 1× non-essential amino acids, 0.14 mM β-mercaptoethanol, 1× penicillin/streptomycin, and Fungizone. Myoblasts were maintained with 10 ng/mL bfgf and then differentiated in DMEM-F12, 2% FBS, 1× insulin–transferrin–selenium, when 80% confluency was reached.

The differentiation method was modified from a previous published method [31 (link)]. The iPSCs were treated with accutase for 2–5 min and harvested by scraping when they reached to 80% confluency. A total of 6000 dissociated iPSCs were then transferred to AggreWell 800 (Stemcell, BC, Canada) dishes and cultivated with gfCDM containing 20 ng/mL bFGF (233-FB, R&D, Minneapolis, MN, USA) for 10 days to form embryoid bodies (EBs). At day 11, the EBs were then transferred to laminin (5 μg/mL; 11243217001, Sigma, SL, USA) and poly-D-lysine (50 μg/mL; A-003-E, Sigma)-coated cell culture dishes and cultivated with gfCDM containing 20 ng/mL bFGF (233-FB/CF, R&D) for 25 days to form Purkinje progenitor cells. During the procedure of EB formation and Purkinje progenitor cells differentiation, the cell culture medium was replaced every other day.

Upon FACS sorting, P75⁺cKIT⁺ melanoblasts were plated onto dried PO/Lam/FN dishes. Cells were fed with melanocyte medium every 2–3 days. Cells were passaged once a week at a ratio of 1:6, using accutase for 20 min at 37 °C for cell detachment. Mature melanocytes at day 100 were used for the seahorse experiments.
Melanocyte media (~1 L):Neurobasal media 500 ml (gibco, 21103-049)
DMEM/F12 500 ml (gibco, 11330-032)
SCF 25 ng/ml (R&D, 255-SC-MTO)
cAMP 250uM (Sigma, D0627)
FGF2 5 ng/ml (R&D, 233-FB/CF)
CHIR 1.5uM (R&D, 4423)
BMP4 12.5 ng/ml (R&D, 314-BP)
EDN3 50 nM (Bachem, 4095915.1000)
25 ml FBS (R&D, S11150H)
2.5 ml penicillin/streptomycin (gibco, 15140-122)
2 ml L-Glutamine (gibco, 25030-081)
B27 supplement (gibco, 12587-010)
N2 supplement (gibco, 17502-048)