Trypsin edta solution

Chemicals such as Dulbecco’s Modified Eagle’s Medium (DMEM), l-Glutamine, Fetal Bovine Serum (FBS), Trypsin-EDTA solution, Antibiotic-Antimycotic solution (10000 U/mL Penicillin, 10 mg/mL Streptomycin and 25 μg/mL Amphotericin B in 0.9 % normal saline for 100 X), MTT dye, Dimethyl Sulfoxide (DMSO), Trypan blue, Dulbecco’s Phosphate Buffered Saline (DPBS), Tris-EDTA, Propidium iodide (PI), Ribonuclease A (RNase A) and Triton X-100 were purchased from Himedia, India. Molecular probes, Fluorescein isothiocyanate-Phalloidin (FITC-Phalloidin), and 4′,6- diamidino-2-phenylindole (DAPI) were obtained from ThermoFisher Scientific, USA. DCFDA, 3,3′-dihexyloxacarcocyanine iodide (DiOC₆), Rotenone, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, disodium phosphate (Na₂HPO₄) 25 % (v/v) glutaraldehyde, paraformaldehyde, osmium tetroxide, uranyl acetate, and lead citrate were purchased from Sigma Aldrich, USA. The kits used in this study such as the SOD Assay kit and Epoxy Embedding Medium Kit were procured from Sigma Aldrich, USA (Cat. No.:19106 and Cat. No.: 45359-1EA-F respectively), and the ATP Determination kit was purchased from Thermo Scientific, USA (Cat. No.: A22066). The absolute ethanol used in this study is of HPLC grade (Commercial Alcohols, Greenfield Global, Canada). All the reagents and chemicals are of analytical grade.

Shilajit was purchased from Yucca Enterprises, India. Zinc acetate was procured from Sisco Research Laboratories Pvt. Ltd. (SRL), India and 0.45 μm filter papers were purchased from Millipore. Isopropanol of analytical grade was purchased from Thermo Fisher Scientific India Pvt. Ltd. All other solvents and chemicals used were of analytical grade. The cisplatin was obtained from the local medical store. Dulbecco’s Modified Eagles Medium (DMEM), Fetal Bovine Serum (FBS), antibiotic and anti-mycotic preparations for cell culture, Trypsin EDTA solution and 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium bromide) (MTT) were obtained from Hi Media Laboratories Pvt. Ltd., India.

Human bone marrow mesenchymal stem cell line (BMSC) was purchased from AddexBio Technologies, CA, USA. Upon receiving the cells in liquid nitrogen, they were immediately washed with phosphate buffered saline (PBS, Gibco, USA) to ensure that the cells were free from the cryoprotectant Dimethyl Sulfoxide (DMSO). The washed cells were then cultured in mesenchymal stem cell growth media (MSCM, AddexBio Technologies, CA, USA) supplemented with 10% Fetal Bovine Serum (FBS, Himedia, USA) and 1% penicillin streptomycin antibiotic solution (Himedia, USA). After the addition of MSCM, the cells were kept in an incubator supplied at 37°C temperature and 5% CO2. The cell culture medium was replaced with fresh medium at an interval of three days, and their confluency appraised. Once the cells achieved 90% confluency, the proteolytic enzyme trypsin was added to dislodge the adherent cells from the culture flask. Trypsinization was done using trypsin/EDTA solution (Himedia, USA). The detached cells were sub-cultured for further growth in MSCM.

Cells were washed with 1X PBS, detached with the trypsin/EDTA solution (Himedia, USA) at 37°C, and centrifuged at 500 g for 5 min at 4°C. The cells were re-suspended in 1X PBS and a 500 μL suspension prepared, with a concentration of 1X10⁶ cells/mL. The suspension was incubated for 30 min at room temperature and divided into tubes. For the determination of RANK and RANKL, one served as negative control to which a mouse IgG (15 μg/mL; Chemicon International, USA) for RANKL and a mouse IgG coupled to phycoerythrin (IgG-PE: 20 μg/ml; R&D Systems) for RANK was added, and the other was labelled with either a mouse anti-human RANKL antibody (15 μg/ml; R&D Systems) or a mouse anti-human RANK-PE (20 μg/ml; Abcam) for 30 min at 4°C. For RANKL, after this period, cells were washed, and a goat anti-mouse FITC-conjugated secondary antibody (7.5 μg/ml; R&D Systems) was added for another 30 min at 4°C. Cells were then re-suspended in PBS and analyzed by using a flow cytometry apparatus (Facscalibur; BD Biosciences, Canada). The control sample was used to determine the background fluorescence and compared with that of the sample incubated with the specific antibody.

Silk cocoons were obtained from the Government Silk Cocoon market, Vellore, India. DNase, RNase, phosphate buffered saline, sodium dodecyl sulfate, Dulbecco’s modified eagle’s medium (DMEM), trypsin—EDTA solution, fetal bovine serum, antibiotic antimycotic solution, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, dimethyl sulphoxide, Luria bertani broth, nutrient agar, and formalin, were purchased from HiMedia Laboratories Private Limited, India. Escherichia coli was procured from Microbial Type Culture Collection, Institute of Microbial Technology, Chandigarh, India.

Carboxymethyl cellulose (CM Cellulose), Dulbecco’s modified eagle medium (DMEM), Fetal bovine serum (FBS), Penicillin, Streptomycin and Trypsin-EDTA Solution were purchased from Himedia (India). Thiazolyl Blue Tetrazolium Bromide (MTT), Dimethyl Sulfoxide (DMSO), Neural Red (NR), Formaldehyde, β-Nicotinamide adenine dinucleotide reduced dipotassium salt (β-NADH), Trypan Blue and Triton X-100 dye solution were purchased from Sigma-Aldrich (USA).