Ham s f10 medium

Satellite cells were isolated from the hind limbs of 4- to 12-week-old male C57BL/6N mice according to the method of Rando and Blau (14 (link)), with modifications. Hind limb muscles were collected, rinsed in PBS, and minced in PBS supplemented with 5 mM CaCl₂, 5 mM collagenase, and Dispase I (400 protease units per milliliter). The minced tissue was incubated at 37°C and triturated every 30 min. After 90 min, the digested slurry was diluted with PBS and passed through 100 μm and then 40 μm cell strainers (Falcon). The filtrate, containing cells and tissue debris, was centrifuged at 350 g for 15 min and the pellet was resuspended in growth medium consisting of Ham’s F-10 medium (Gibco) supplemented with 20% heat-inactivated FBS, 1% antibiotic antimycotic (Sigma), and 5 ng/ml basic fibroblast growth factor (R&D Systems). The cells were cultured on petri dishes (Rikaken; RSU-SD9015-2), which had been coated with type I collagen (Sigma; C8919). Culture media were replaced every 2–3 days, and cells were maintained at less than 70% confluency. Myoblasts were enriched during passaging by selective detachment in PBS and preplating to remove contaminating fibroblasts (15 (link)). Satellite cells were used for experiments within 10 passages.

Transcription levels were measured by pulse labeling with the nucleotide analogue 5′ethynyl uridine (EU) (Jena Bioscience). Cells were grown to 80% confluency on glass coverslips, treated as indicated, and incubated for 30 min with 1 µM EU in Ham’s F10 medium supplemented with 10% dialyzed fetal calf serum (Gibco). Subsequently, cells were washed with PBS, fixed with 2% PFA in PBS for 15 min. After permeabilisation with 0.1% triton in PBS for 10 min, click chemistry-based azide coupling was performed by incubation for 30 min with 60 µM Atto594 Azide (Attotec, Germany) in 50 mM Tris buffer (pH 8) with 4 mM CuSO₄ (Sigma Aldrich) and 10 mM freshly prepared ascorbic acid. Coverslips were washed with PBS and mounted with Vectashield containing DAPI (Brunschwig Chemie). All steps were performed at RT. Cells were imaged with a Zeiss LSM 700 Axio Imager Z2 upright microscope equipped with a 63x Plan-Apochromat oil immersion lens (NA 1.40) using Carl Zeiss LSM (version 14.0.0.0) and image analysis was performed using Image J.

At 20 h post transduction, virus supernatant fraction was removed from HLA-A02:01 transduced cells by centrifugation. The cells were cultured in Ham’s F-10 medium (Gibco) supplemented with 200 mmol/l glutamine (Gibco), 0.5% BSA fraction V (Sigma-Aldrich, Zwijndrecht, the Netherlands), 1 mol/l CaCl₂ and 1.8 g/l d-glucose for 4 days to allow transgene expression before trypsin dispersion and incubation with PPI-specific CTL clone 1E6 or CMV-specific clone 18 in a 1:5 ratio. After overnight incubation, cells were stained with Fixable Viability Dye eFluor 450 (eBioscience, Vienna, Austria), CD45-PerCP (2D1; BD, Breda, the Netherlands) and HLA-A2–fluorescein isothiocyanate (FITC) (BD), then fixed in paraformaldehyde and permeabilised with saponin before staining with guinea pig anti-insulin (Free University Brussels, Brussels, Belgium) and donkey anti-guinea pig Alexa 647 (Jackson Immunoresearch Laboratories, West Grove, PA, USA). Cells were measured on a CANTO II flow cytometer (BD). Transduction efficiency was determined by staining with anti-HLA-A2–Allophycocyanin (APC) (Bb7.2; BD) on a Calibur flow cytometer (BD).

GH3 cells were obtained from the Institute of Basic Medical Sciences of the Chinese Academy of Medical Sciences (Beijing, China) and were maintained in Ham’s F-10 medium (Gibco-BRL, Carlsbad, CA, USA) containing 12.5% horse serum (Gibco-BRL), 2.5% HyClone^™ fetal bovine serum (ThermoFisher Scientific, Waltham, MA, USA), 2 mmol/l L-glutamine (Sigma), 0.25 μg/ml Fungizone^® (Invitrogen Life Technologies) and 80 μg/ml gentamicin (Sigma). The cells were plated at various densities and were incubated for four days in the maintenance medium. Prior to treatment, the medium was replaced with the treatment medium, a defined serum-free, phenol red-free medium, containing Ham’s F-12 medium (Gibco-BRL) with 10 μg/ml insulin (Sigma), 5 μg/ml transferrin (Sigma) and 0.5 ng/ml parathyroid hormone (Sigma). After 24 h, cells were treated with or without E2 and RE at various concentrations for different time periods.