After collecting the NP specimens during surgery, we transferred the tissue to a clean bench using a sterile centrifuge tube and rinsed it three times with phosphate-buffered saline (PBS, Cytiva). Next, the NP tissue was digested with type II collagenase (0.2%, Sigma-Aldrich) for 4 h at 37°C. After centrifugation, the supernatant was removed, and the tissue was resuspended and cultured in DMEM/F12 medium (Corning) with 10% fetal bovine serum (FBS, SANTACRUZ). Adherent NP cells were observed under a microscope after five days of culture without replacing the medium. When the cell density reached 80–90%, we passaged the NP cells using Trypsin-EDTA (Biosharp), and the passaged NP cells were the first passage. Only NP cells within four passages were used for in vitro studies.
Trypsin edta
Manufactured by Biosharp
Sourced in China
Trypsin-EDTA is a solution used for the dissociation and detachment of adherent cells in cell culture. It contains the enzyme trypsin, which breaks down the proteins that hold cells together, and EDTA, which chelates calcium and magnesium ions necessary for cell-cell adhesion. This solution is commonly used to passagecells and prepare single-cell suspensions for various cell-based applications.
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Lab products found in correlation
Collagenase type 2, by Merck Group (2 mentions)
Dmem f12 medium, by Corning (1 mentions)
Fetal bovine serum (fbs), by Santa Cruz Biotechnology (1 mentions)
Phosphate buffered saline (pbs), by Cytiva (1 mentions)
Total rna isolation kit, by Vazyme (1 mentions)
Cck 8 kit, by AbMole (1 mentions)
Hiscribe t7 in vitro transcription kit, by New England Biolabs (1 mentions)
Rnase h, by New England Biolabs (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Chamq universal sybr qpcr master mix, by Vazyme (1 mentions)
Hiscript 3 rt supermix for qpcr, by Vazyme (1 mentions)
Penicillin streptomycin, by Biosharp (1 mentions)
Mem medium, by Thermo Fisher Scientific (1 mentions)
Neurobasal medium, by Thermo Fisher Scientific (1 mentions)
Osteogenic medium, by Cyagen (1 mentions)
Penicillin streptomycin double antibody, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium high glucose dmem hg, by Thermo Fisher Scientific (1 mentions)
Alizarin red stain, by Cyagen (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
8 protocols using trypsin edta
1
Isolation and culture of NP cells
2
Transfection of 293T Cells with pEGFP-C1
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293T cell lines and pEGFP-C1 plasmids were derived from our laboratory. Growth media (DMEM) was supplemented with 10% FBS and 1% antibiotic–antimycotic. 293T cell lines were maintained up to 30 passages in a 37 °C/5% CO2 incubator. All oligonucleotides were synthesized by Sangon (Shanghai, China). Trypsin EDTA and penicillin streptomycin were purchased from Biosharp (Hefei, China). DMEM basic media and fetal bovine serum and Opti-MEM were purchased from Gibco. Lipofectamine 3000 was purchased from Invitrogen. A CCK-8 kit was purchased from AbMole. A HiScribe™ T7 in vitro Transcription Kit and RNase H were purchased from NEB. A Total RNA Isolation Kit, HiScript III RT SuperMix for qPCR, and ChamQ Universal SYBR qPCR Master Mix were purchased from Vazyme (Nanjing, China).
3
Isolation and Culture of Human NP Cells
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Human NP tissue was isolated from the surrounding fibrous tissue and washed with sterile phosphate-buffered saline (PBS, Sigma) before mincing into approximately 0.5 × 0.5 × 0.5 mm3 fragments using sterile ophthalmic scissors. The fragments were digested with a 2 mg/mL solution of type II collagenase (Sigma-Aldrich, USA) at 37℃ for 6 h, followed by centrifugation at 2000 rpm for 5 min. Cell pellets were resuspended and cultured in DMEM medium (HyClone, USA) for 24 h to remove non-adherent and non-viable cells. The isolates were passaged weekly using 0.25% trypsin–EDTA (Biosharp), with cells from the second passage (P2) utilized for the in vitro experiments. The culture medium was replaced with acid-conditioned medium prepared as previously described [5 (link)], or serum-free DMEM for 12 h before treatment with 0–6 μg/mL recombinant human COMP (rhCOMP; R&D Systems, Minneapolis, MN).
4
Culturing Hippocampal Neurons for Ca2+ Analysis
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The protocol for culture of hippocampal neurons was based on a standard procedure with minor modifications 193 (link), 194 . In brief, isolated hippocampus was chopped into small pieces in MEM medium (Gibco Life Technologies, Grand Island, USA) and then digested with 5 mL 0.25% trypsin-EDTA (biosharp, Hefei, PR China) at 37°C for 30 min. The digested tissue was further ground in 70 µm cell strainer to obtain the single cell suspension. After centrifugation at 2000 rpm for 2 min, the cell pellet was washed once with phosphate buffered saline (PBS) buffer. Cells were then resuspended in appropriate volume of plating medium (MEM supplemented with 10% horse serum, 100 U/mL penicillin and 100 µg/mL streptomycin) and seeded in 12 well plate containing poly-L-lysine (PLL, Merck, Darmstadt, Germany) coated coverslips. After incubation at 37°C and 5% CO2 for several h, when the cells having attached to the coverslips, the plating medium was replaced with complete Neurobasal medium (Neurobasal medium (Gibco) supplemented with 2% B27 (Gibco), 0.5 mM L-glutamine and antibiotics), and the cells were further incubated at 37°C and 5% CO2 for 1 or 2 weeks for Ca2+ detection and PLA.
5
Osteogenic Differentiation of MC3T3-E1 Cells
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CS (medium molecular weight) and β-GP were purchased from Sigma-Aldrich (USA), QCS with degree of substitution 90 % and nHA were purchased from Aladdin Co., Ltd. (Shanghai, PR China), and phosphate-buffered saline (PBS) and 4 % paraformaldehyde were purchased from Solarbio (Beijing, PR China). High-glucose Dulbecco's Modified Eagle's Medium (HG-DMEM) and penicillin streptomycin double antibody were purchased from Gibco (Grand Island, NY, USA). Fetal bovine serum (FBS) was purchased from SenBeiJia Biological Technology Co., Ltd. (Nanjing, PR China). Trypsin-EDTA (0.05 % trypsin and 0.02 % EDTA) solution was purchased from Biosharp (Beijing, PR China). Cell Counting Kit-8 (CCK-8), calcein-AM-PI staining kit, RIPA lysis buffer, BCIP/NBT alkaline phosphatase (ALP) color development kit, and ALP assay kit were purchased from Beyotime Co., Ltd. (Shanghai, China). Osteogenic medium of mouse embryo osteoblast precursor cells (MC3T3-E1) and alizarin red stain were purchased from Cyagen (Santa Clara, USA). A FastPure Cell/Tissue Total RNA Isolation Kit, ABScript Ⅲ RT Master Mix for qPCR with gDNA Remover, and Universal SYBR Green Fast qPCR Mix were obtained from Abclonal Co., Ltd. (Wuhan, PR China).
6
Oleanolic Acid Extraction and Cell Evaluation
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Oleanolic acid was extracted from Ligustrum lucidum by ultrasonic-assisted technology. Dulbecco’s modified Eagle’s medium (DMEM) was obtained from biological industries (Shanghai, China). A CCK-8 kit, nonessential amino acids (NEAAs), trypsin−EDTA, PBS and penicillin−streptomycin (PS) were obtained from Biosharp (Hefei, China). Cell cycle and apoptosis analysis kits were obtained from Beyotime (Shanghai, China). An Annexin V-FITC/PI apoptosis detection kit was obtained from Vazyme (Nanjing, China).
7
Isolation and Culture of Human Annulus Fibrosus Cells
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Human NP tissue was isolated from peripheral annulus fibrous tissue and washed 3 times with sterile phosphate buffered saline (PBS, Sigma), minced carefully with aseptic ophthalmic scissors into ~0.5×0.5×0.5 mm pieces. A proportion of the minced pieces were reserved for the preparation of freshly isolated tissue homogenates while the rest were digested in 2 mg/ml type II collagenase solution (Sigma-Aldrich, USA) at 37° C for 4 hours. Following 1000×g centrifugation for 5 min, pellets (liberated cells and partially digested tissues) were collected and cultured with low-glucose Dulbecco’s modified Eagle’s medium (2 mM L-glutamine, HyClone) supplemented with 10% FBS, 1% penicillin/streptomycin in a humidified incubator at 37° C with 5% CO2. Culture medium was replaced twice a week with cells trypsinized with 0.25% Trypsin-EDTA (Biosharp) for further culture upon 80-90% confluence with subculture at 1:3 as passage 1 (P1). Cells at passage 2 were used for the in vitro experiments.
8
Quantifying Cardiomyocyte Proliferation
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ES-CMs clusters were collected at day 10 of differentiation, and dissociated into single cells using 0.05% Trypsin/EDTA (Biosharp). 5×10 4 to 1×10 5 cells per well were seeded in a gelatin-coated 24-well-plate with or without 100 µM puerarin supplemented with 10 µM EdU (5-Ethynyl-2'-deoxyuridine) from the Click-iT® EdU Imaging Kits C10338 (Invitrogen). After 48 h or 72 h cultivation, the staining of ES-CMs with EdU was performed according to the instructions of the manufacturer. ES-CMs were identified by staining with mouse IgG anti-αsarcomeric-actinin antibody (1:200, Sigma) overnight at 4°C. The secondary goat anti-mouse FITC-conjugated IgG (1:200, Proteintec) was used for 1 h at room temperature. Nuclei were stained with Hoechst33342 (5µg/mL, Invitrogen). Images were taken randomly using a fluorescence microscope (Nikon, TE2000-S). The total number of ES-CMs was determined by counting α-actinin/Hoechst33342 double positive cells. The percentage of proliferating ES-CMs was determined by dividing the number of α-actinin/EdU/ Hoechst33342 triple positive cells by the total number of ES-CMs. The counting was carried out independently by three operators. The proliferation assays were performed by 5 to 6 independent experiments, and total 3002 of control cells and 3166 of puerarin treated cells were counted.
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