Atcc crl 1772

The C2C12 cell line (ATCC® CRL1772™, ATCC®, Manassas, Virginia, USA) was used. This is a subclone of the mouse myoblast cell line established by D. Yaffe and O. Saxel^{53 (link)} and was already used for in vitro evaluation of implantable muscular tissue scaffolds^{54 (link)} or bioeffects induced by physical therapies such as ultrasound.^{55 (link)} The biological response of the three magnet coatings and of the uncoated magnets (control condition) was evaluated both qualitatively using fluorescence and bright field microscopy analyses, and quantitatively using WST-1 cell metabolism assay and DNA content assessment with the PicoGreen kit. The data collected through WST-1 and PicoGreen assays were statistically evaluated using the Kruskal–Wallis post-hoc test with Dunn's multiple comparison test in order to identify significant differences among the coatings under investigation. Results were considered statistically different for p-values ≤0.05.

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test was performed using the C2C12 myoblast cell line (ATCC^® CRL-1772™; (ATCC, Manassas, VA, USA). Briefly, Polysaccharide conjugates A–C with peptides 1–3 at (2 cm² of modified non-woven) were incubated in DMEM (2 mL) (Corning) for 48 h at 37 °C to attain extracts. Then, cells were seeded in a 96-well plate at a density of 1 × 10⁴ cells/well and grown at 37 °C (5% CO₂). At 24 h, the culture medium was replaced by extract solutions. After 24 or 72 h, the culture medium was removed, and 1 mg/mL of MTT was added. Following a 2 h incubation, the MTT solution was discarded, and 100 µL of isopropanol (Sigma Aldrich, Poznan, Poland) was added. The absorbance of the culture was read at 570 and 650 nm using a microplate reader (Victor X4 Perkin Elmer; PerkinElmer, Inc., Waltham, MA, USA). Cells grown for 48 h in DMEM were used as a negative control, while those treated with dimethyl sulfoxide (DMSO) were used as a positive control. All experiments were performed in triplicate. The results are expressed as a percentage of the negative control with the standard deviation (SD). Statistical significance was assessed using a one-way analysis of variance (ANOVA) and assumed at *** p < 0.001, ** p < 0.01, and * p < 0.05.

The C2C12 cell line (ATCC^® CRL-1772™) from the ATCC collection (Manassas, VA, USA) was used for the analysis. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and antibiotics (50 U/mL of penicillin and 50 µg/mL of streptomycin; Life Technologies, Warsaw, Poland) under aseptic-standard conditions (37 °C, 95% humidity, 5% CO₂). Cells were grown in continuous cultures and passaged 4–12 times with 0.25% trypsin (Life Technologies, Warsaw, Poland) after reaching 80–90% confluence. Then, cells were thawed from the cell bank and passaged twice. Following trypsinization, cells were centrifuged at 400× g for 5 min. The resulting cell pellets were resuspended in 1 mL of fresh media and counted (ADAM MC Automated Mammalian Cell Counter; Digital Bio, Seoul, Korea).

RMS cell lines (CW9019, RD, and RH4) were kindly provided by Dr. Oscar Martínez-Tirado and Dr. Cristina Muñoz-Pinedo from the Bellvitge Biomedical Research Institute (IDIBELL). C2C12 cells were purchased at ATCC (ATCC^® CRL-1772^™, ATCC, Manassas, VI, USA). Human skeletal muscle myoblasts (HSMM) were purchased at Lonza (CC-2580, Lonza, Basel, Switzerland). RMS cell lines were cultured in RPMI 1640 medium (31870, Thermo Fisher, Gibco, Paisley, Scotland) supplemented with 10% heat-inactivated fetal bovine serum (10270, Thermo Fisher, Gibco), 1% of l-glutamine (25030, Thermo Fisher, Gibco), and 1% of penicillin and streptomycin (15140, Thermo Fisher, Gibco). C2C12 cells were cultured in DMEM high glucose medium (41965, Thermo Fisher, Gibco) supplemented with 10% heat-inactivated fetal bovine serum (10270, Thermo Fisher, Gibco) and 1% of penicillin and streptomycin (15140, Thermo Fisher, Gibco). HSMM cells were cultured in SKBM-2 medium (CC-3246, Lonza) supplemented with its specific SingleQuots^TM and growth factors (CC-3244, Lonza). All cells were tested for mycoplasma and maintained at 37 °C in a humidified atmosphere of 5% CO₂. Drug treatments were performed directly in the culture media at the doses and time points indicated in every single experiment. All drugs were purchased at Selleckchem (Munich, Germany).

Murine myoblasts (C2C12, ATCC^® CRL‐1772™) were purchased from ATCC (Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (P/S) for 3 days. Cells were grown in 10 cm dish (for measurement of N^τ‐methylhistidine release and protein content) or 6‐well plate (for all other experiments) at 37 °C in 5% (v/v) CO₂ in a humidified environment. To induce myotube formation, the medium was replaced with DMEM supplemented with 2% horse serum and 1% P/S for 5 days. All drugs were made up in phosphate‐buffered saline (PBS) and subsequently added to the appropriate tissue culture wells (1 : 1000). The vehicle‐control wells received just PBS in the same volume as the treatment groups. To study the effects of β₂‐AR agonist treatment, C2C12 myotubes were treated with clenbuterol (1 μm), because this concentration of clenbuterol has been reported to successfully decrease Atrogin‐1/MAFbx mRNA expression in C2C12 myotubes [28 (link)]. In addition, an inhibitor of adenylyl cyclase (SQ22536, 10 μm) was used to block cAMP synthesis.

Colon-26 (C26) adenocarcinoma cells (kindly provided by Dr. D.O. McCarthy, Ohio State University, Columbus, OH, USA) were maintained in RPMI 1640 (Westburg BV, Leusden, The Netherlands) supplemented with 10% foetal bovine serum (FBS, Sigma-Aldrich, St. Louis, The United States of America), and 1% penicillin/streptomycin (P/S, Fisher Scientific, Waltham, The United States of America) at 37 °C in a 5% carbon dioxide environment. C2C12 muscle myoblast cells were obtained from American Type Culture Collection (ATCC-CRL-1772, ATCC, Manassas, VA, USA) and maintained in growth medium (GM) consisting of DMEM (glutamine)(Westburg BV, Leusden, The Netherlands) supplemented with 10% FBS, and 1% P/S at 37 °C in a 10% carbon dioxide environment. To induce myogenic differentiation, at near-confluence, GM was substituted with differentiation medium (DM) consisting of DMEM supplemented with 2% horse serum (HS) (Fisher Scientific, Waltham, The United States of America), and 1% P/S. DM was routinely changed every 24 h.