Collagenase b

Rat cardiomyoblasts (H9C2 cell line) were maintained in a growth medium with Dulbecco’s Modified Eagle Medium (DMEM) Glutamax supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin at 37 °C in 5% CO₂. The cultured H9C2 cells were grown to 80–90% in a Petri dish and were exposed to EMFs for 24 h, 48 h, and 72 h. Cardiac myocytes were isolated from male 10-week-old C57BL6 mice using the method described by Xu et al. [23 (link)]. Briefly, hearts were perfused by the Langendorff method with HEPES-buffered Earle’s balanced salt solution (GIBCO-BRL) supplemented with 6 mM glucose, amino acids and vitamins (buffer A), and then with buffer A containing 0.8 mg/mL collagenase B (Boehringer-Mannheim, Mannheim, Germany) and 10 μM CaCl₂. The enzyme solutions were filtered (2-μm pores) and recirculated through the heart until the flow rate doubled (12–20 min); the left ventricle was then removed and minced in collagenase containing buffer A. Thereafter, the tissue pieces were transferred to fresh (enzyme free) buffer A supplemented with 1.25 mg/mL taurine, 5 mg/mL BSA (Sigma-Aldrich, St. Louis, MO, USA), and 150 μM CaCl₂ and mechanically dissociated by gentle trituration. The resulting suspension was filtered and isolated cells were obtained by sedimentation. The cultured primary cardiomyocytes were exposed to 915 MHz EMFs for 1 h, 3 h, and 24 h.

Normal articular cartilage was collected from five white New Zealand buck rabbits, which were anesthetized with overdose of propofol and euthanized using Potassium chlorate 60 mg/Kg. After knee joint surgery, the pieces of articular cartilage were dissected and separated from the underlying bone and connective tissues. The pieces were washed three times with PBS 1× and 5% penicillin/streptomicin. The extracted cartilage was digested in a solution of 2 mg/mL Protease Type XIV. Bacterial from (Streptomyces griseus) (Sigma Aldrich, St Louis, MO, USA) in PBS 1× for 1.5 h and 1.5 mg/mL collagenase B (Roche, Meylan, France) in basic medium DMEM at 37 °C overnight. This suspension was centrifuged at 1200 rpm for 8 min. to collect the chondrocytes and was washed with basic medium DMEM. The chondrocytes were cultured in DMEM/F12 (1:1 with 15% FBS plus 1% antibiotic mixture of penicillin/streptomycin) at a density of 1 × 10⁵ cells/mL and incubated in a humidified atmosphere of 5% CO₂ at 37 °C. Culture medium was changed every two days and each passage was made when the confluence reached between 80–90%. We only used the second passage of cells in all experiments in order to avoid loss of chondrocyte phenotype [92 ].

Lungs from Rptor^f/f; Shh^Cre/+, Tfam^f/f; Shh^Cre/+and Cox10^f/f; Shh^Cre/+ mice and their littermate controls were dissected at the indicated time points, placed in the digestion solution (1.2 U/ml dispase (BD Biosciences, Cat# 354235), 0.5 mg/ml collagenase B (Roche, Cat# 11088815001) and 50 U/ml DNase I (QIAGEN, Cat# 79254)) and rocked at 37 °C for 1 h to release individual lungs cells. An equal volume of culture medium (DMEM (Mediatech, Cat# 10-013-CV) with 10% FBS (Gibco, Cat# 10437-028), 2x penicillin/streptomycin (Gibco, Cat# 15070-063) and 1x L-glutamine (Gibco, Cat# 25030-081)) was added to the digested lung samples, which were filtered through 40 µm cell strainers. After centrifugation at 600 × g for 10 min, the cell pellets were resuspended in 200 µl purification buffer (Phenol red-free DMEM with 0.2% BSA and 2% FBS). Biotin-conjugated EpCAM antibody (anti-CD326 (EpCAM), 1:100, eBioscience, Cat# 13-5791-82; RRID:AB_1659713) and Dynabeads M-280 Streptavidin (Invitrogen, Cat# 11205D) were added to the samples and incubated at 4 °C for 1 h with shaking. Epithelial cells were captured using Dynabeads magnets following the manufacturer’s instructions. Purified lung epithelial cells were used for qPCR, ATP measurement and Western blotting analysis.

Cardiac cultures were dissociated using 0.25% trypsin/EDTA (Gibco/BRL) for 5 min at 37 °C to quickly lift the monolayer cells off the plate, then a single cell suspension was achieved with a solution of collagenase/DNase (PBS with 20% serum, 10 mg ml⁻¹ collagenase A (Roche, cat#11088785103), 10 mg ml⁻¹ collagenase B and 10 μg ml⁻¹ DNase I (Roche, cat# 1184932001), 5–10 min at 37 °C. Cells were stained with live/dead stain (Thermofisher, cat# L34955) for 20 min, fixed in 2% PFA and permeabilised in 0.5% saponin (Sigma, cat# S7900). After a wash in PBS/0.1% saponin, cells were stained with anti-cTNT (Abcam, clone 1C11) primary antibody and Alexa-488 donkey anti-mouse (Thermofisher, cat# A21202) secondary antibody.

Knee cartilage was dissected from newborn WT and ERp57 cKO mice and incubated overnight in DMEM supplemented with 1 mg/mL Collagenase B (Roche, Mannheim, Germany) and 1 mM cysteine. Next, 400,000 of these isolated cells were pipetted as 20 µL droplets of cells in DMEM to form micromass cultures in a 24-well plate. After 3 h, 1 mL DMEM complete supplemented with ABCP was added to the micromass cultures, which were then cultivated for 7 days at 37 °C and 5% CO₂. Micromasses were then fixed overnight with 2% (v/v) PFA and 2.5% (v/v) glutaraldehyde in 100 mM cacodylate buffer pH 7.4 at 4 °C. Micromasses were washed in PBS and postfixed in 0.5% (v/v) osmium tetroxide and 1% (w/v) potassium hexacyanoferrate (III) in 0.1 M cacodylate buffer at 4 °C for 2 h. After another washing step with distilled water, the samples were dehydrated in an ascending ethanol series and subsequently incubated in propylene oxide (2 × 15 min). Micromasses were embedded in Epon and ultrathin sections were cut with an ultramicrotome. Sections were collected on copper grids and negatively stained with 2% uranyl acetate for 10 min. A Phillips EM-410 electron microscope was used to take electron micrographs at 60 kV using imaging plates (Ditabis, Pforzheim, Germany).