MG-63 human osteoblast-like cells (KCLB No. 21427) were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea), and primary gingival fibroblasts (PCS-201-018) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were seeded on 10-cm tissue culture dishes in standard cell culture medium consisting of Dulbecco’s modified Eagle’s medium (Invitrogen, Waltham, MA, USA) containing 10% foetal bovine serum (Invitrogen) and antibiotics (50 U/mL penicillin G and 50 μg/mL streptomycin; Invitrogen). Samples were incubated at 37 °C in a 5% CO2/95% air atmosphere at 100% relative humidity. The cell culture medium was changed every 2–3 days. When the cells reached 85–90% confluence, they were treated with a trypsin-ethylenediaminetetraacetic acid solution (Invitrogen), resuspended in culture medium, and seeded in new dishes at a 1/5 dilution.
Trypsin ethylenediaminetetraacetic acid solution
Manufactured by Thermo Fisher Scientific
Sourced in United States, Japan
Trypsin-ethylenediaminetetraacetic acid (Trypsin-EDTA) solution is a laboratory reagent used for cell detachment and dissociation. It contains the proteolytic enzyme trypsin and the chelating agent EDTA, which together facilitate the disruption of cell-cell and cell-matrix adhesions, enabling the harvesting of adherent cells from culture vessels.
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Lab products found in correlation
Penicillin streptomycin solution, by Thermo Fisher Scientific (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Antibiotic antimycotic solution, by Thermo Fisher Scientific (4 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (3 mentions)
Nano3 anhydrous ethanol, by Merck Group (3 mentions)
H2so4, by Merck Group (3 mentions)
Kmno4, by Merck Group (3 mentions)
Sodium hydroxide (naoh), by Merck Group (3 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (2 mentions)
Nitric acid solution, by Fujifilm (2 mentions)
Nitric acid solution, by Hitachi (2 mentions)
Icap6000 emission spectrometer, by Hitachi (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Dulbecco s modified eagle s medium, by Merck Group (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (2 mentions)
Gt powder, by Merck Group (2 mentions)
In vitro toxicology assay kit, by Merck Group (2 mentions)
H2o2 aqueous solution, by Merck Group (2 mentions)
26 protocols using trypsin ethylenediaminetetraacetic acid solution
1
Culturing MG-63 Osteoblast-like and Primary Gingival Fibroblast Cells
2
DMEM-Based Cell Culture Protocols
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Dulbecco's modified Eagle's medium (DMEM) was purchased from WelGENE Inc. (Daegu, Korea). The adenosine, L-DOPA, 1-phenyl-2-thiourea, and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibiotic-antimycotic, fetal bovine serum (FBS) and trypsin-ethylenediaminetetraacetic acid solution were obtained from Gibco-Invitrogen (Carlsbad, CA, USA).
3
Isolation and Culture of Adipocytes
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Preparation of IFP- and SC-DFATs using the ceiling culture method was described previously [23 (link)]. Briefly, approximately 1–2 g of SC and IFP fat tissue was minced and digested with 0.1% (W/V) type II collagenase solution (Sigma-Aldrich, St. Louis, MO) at 37 °C for 30 min. After filtration and centrifugation at 135×g for 1 min, the floating top layer containing unilocular adipocytes was collected. The cells were washed with phosphate-buffered saline (PBS), placed in a T-12.5 cell culture flask (NUNC, Roskilde, Denmark) filled with Dulbecco's Modified Eagle's Medium (DMEM, Invitrogen, Carlsbad, CA) containing 20% fetal bovine serum (FBS, Lot 6G2146; JRH Biosciences Inc., Lenexa, KS), and incubated at 37 °C under 5% CO2. The cells floated up and adhered to the top inner ceiling surface of the flask. At 7 days after culturing, the medium was removed and the flasks were turned upside-down so that the cells were oriented on the bottom. The medium was exchanged every 3 or 4 days until the cells reached confluency. For passage, the cells were harvested by treating the cells with a trypsin-ethylenediaminetetraacetic acid solution (Invitrogen), following which the cells were seeded in 100-mm dishes at a density of 1 × 106 cells per dish and cultured. Cells at the second passage (P2) were used in the experiments.
4
Culturing Neural Stem Cells and Cancer Cell Lines
5
Boron Uptake Efficiency of TPFC in F98 Cells
6
Synthesis and Cytotoxicity Evaluation
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Ammonium sulphate, zirconium oxy nitrate, and ammonium hydroxide (28%–30%) were obtained from Sigma-Aldrich (St Louis, MO, USA). A trypsin/ethylenediamine tetraacetic acid solution was purchased from Invitrogen (Carlsbad, CA, USA). Dimethylsulfoxide, phosphate-buffered saline, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and Dulbecco’s Modified Eagle’s Medium were purchased from Sigma-Aldrich.
7
Expansion and Characterization of hMSCs
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Frozen hMSCs from passage 0 to 2 were delivered from the Tulane Center for Gene Therapy. hMSCs isolated from the bone marrow of multiple de-identified healthy donors from age 19–49 years (Table S1 ) were collected from plastic adherence, with less than 2% expression of CD3, CD14, CD31, CD45 and CD117, greater than 95% expression of CD73, CD90, CD105 and CD147, while simultaneously possessing tri-lineage differentiation potential upon in vitro induction [36 (link)]. hMSCs (1 × 106 cells/mL/vial) were cryopreserved in a culture media encompassing α-MEM, 2 mM L-glutamine, 30% fetal bovine serum (FBS) and 5% dimethyl sulfoxide (DMSO). hMSCs were thawed and seeded at 2500 cells/cm2 in a complete culture media containing α-MEM, 10% FBS (Atlanta Biologicals, Lawrenceville, GA, USA), and 1% penicillin/streptomycin (Life Technologies, Carlsbad, CA, USA) in a standard culture incubator at 37 °C at 5% CO2. The culture medium was changed every two days. Cells were grown to 80% confluence and harvested with a 0.25% trypsin/ethylenediaminetetraacetic acid solution (Invitrogen, Grand Island, NY, USA) at 37 °C for 5–7 min. Harvested cells were re-plated at a density of 2500 cells/cm2 and sub-cultured up to passage 4–6 for experiments.
8
Integrin-mediated cell adhesion protocol
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GO powder was provided by Yongjun Gao's Group of Hebei University. Methoxypolyethylene glycol (mPEG‐NH2, Mw = 2000) was purchased from Beijing JenKem Technology. 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide (EDC) was purchased from Beijing J&K China Chemical Ltd. Sodium chloroacetate was purchased from Shanghai Sigma‐Addiction. Dulbecco's modified Eagle's medium (DMEM), penicillin/streptomycin solution, trypsin‐ethylenediaminetetraacetic acid solution, phosphate buffer saline (PBS) were all acquired from Invitrogen. Radioimmunoprecipitation assay buffer (high), 4% paraformaldehyde, and 5% bovine serum albumin (BSA) blocking buffer were purchased from Solarbio Life Science. Membrane and Cytosol Protein Extraction Kit was purchased from Beyotime. ITGB8 antibody, ITGAV antibody, GADPH antibody, and Talin‐1 antibody were all purchased from eBioscience. The chemicals above were all analytical grade. Alexa Fluor 647 goat anti‐mouse IgG (H+L), Alexa Fluor 488 goat anti‐rabbit IgG (H+L), β‐BODIPY 500/510 C12‐HPC, Micro BCA Protein Assay Kit, and Pierce Co‐Immunoprecipitation Kit were obtained from Thermo Scientific.
9
Expansion and Maintenance of MSCs
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BM-and C-derived MSCs were cultured in 75 cm 2 flasks, re-fed twice a week with 12.5 ml of fresh medium, detached at confluence, usually once a week, by trypsinethylenediaminetetraacetic acid solution (Invitrogen) treatment, counted, partly frozen and partly reseeded for culture at 37 °C in 5% CO 2 /O 2 atmosphere. Each weekly re-seeding or passage (P) was defined by a progressive number.
10
Cellular Boron Uptake Quantification
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In order to investigate the ability of TPFC for delivery of boron intracellularly, a cellular uptake was conducted, in which the cells were exposed to 10 μg 10 B/mL of TPFC or BPA for 2.5, 6, 12, and 18 h. First, F98 rat glioma cells were seeded in 100 mm dishes (BD Falcon ™ , Franklin Lakes, New Jersey), and the culture medium was exchanged for boron compounds-containing (TPFC, BPA) culture medium just before confluence. In this study, three 100 mm dishes for each designated time were used. After the completion of exposure, boron compounds-containing culture medium was removed, and then the cells were washed twice with 4°C phosphate-buffered saline (PBS) and detached with trypsin-ethylenediamine tetraacetic acid solution. Medium was then added, and the cells countered and sedimented (centrifugation: 214.2 G-force for 5 min). Cells were digested overnight with 1 N nitric acid solution, and boron uptake was determined by inductively coupled plasma atomic emission spectrometry (ICP-AES) using an iCAP6000 emission spectrometer (Hitachi High-Technologies, Tokyo, Japan). PBS and trypsin-ethylenediamine tetraacetic acid solution were purchased from Gibco Invitrogen, and the nitric acid solution was purchased from Wako Pure Chemical Industries (Osaka, Japan).
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