CuSO4·5H2O was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Sodium dodecyl sulfonate (SDS), aprotinin, leupeptin, pepstatin A, and phenylmethylsulfonyl fluoride (PMSF) were obtained from AMRESCO Inc. (Solon, OH, USA). Cell Counting Kit-8 (CCK-8) was purchased from Med. Chem. Express (Shanghai, China). Superoxide dismutase (SOD), catalase (CAT), and 4-phenylbutyric acid (4-PBA) (purity ≥ 98%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Life Technologies Corporation (Grand Island, NY, USA). 2′,7′-dichlorofluorescein-diacetate (DCFH-DA) and 0.25% Trypsin-EDTA were purchased from Beyotime Biotechnology (Haimen, China). All other chemicals were of analytical grade.
Trypsin edta
Manufactured by Beyotime
Sourced in China, United States
Trypsin-EDTA is a laboratory reagent used for the dissociation and detachment of adherent cells in cell culture applications. It contains the enzyme trypsin, which cleaves peptide bonds, and EDTA, which chelates divalent cations and aids in the cell dissociation process. This product is commonly used to subculture or passage adherent cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (15 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (5 mentions)
Penicillin streptomycin, by Beyotime (4 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
Ferrous sulfate, by Xilong (3 mentions)
Phosphate buffered saline (pbs), by Beyotime (2 mentions)
Penicillin, by Beyotime (2 mentions)
Streptomycin, by Beyotime (2 mentions)
Phosphate buffered saline (pbs), by Wuhan Servicebio Technology (2 mentions)
Reactive oxygen species assay kit, by Beyotime (2 mentions)
Dulbecco s modified eagle s medium dmem, by Merck Group (2 mentions)
Phosphate buffered saline (pbs), by Solarbio (2 mentions)
4 phenylbutyric acid 4 pba, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Cuso4 5h2o, by Sinopharm (1 mentions)
2 7 dichlorofluorescein diacetate dcfh da, by Beyotime (1 mentions)
Chlorpromazine, by Merck Group (1 mentions)
Nystatin, by Merck Group (1 mentions)
Yoyo 1, by Thermo Fisher Scientific (1 mentions)
Lysotracker red dnd 99, by Thermo Fisher Scientific (1 mentions)
33 protocols using trypsin edta
1
Copper Sulfate Induced Oxidative Stress
2
Plasmid Transfection and Cellular Assays
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Linear PEI25K was purchased from Polysciences (USA). Chlorpromazine, nystatin, and cytochalasin D were purchased from Sigma-Aldrich (USA). Nuc-Cyto-Mem Preparation Kit was purchased from Applygen Technologies (Beijing, China). The plasmids of peGFP-N1 encoding enhanced green fluorescent protein, pRFP encoding red fluorescent protein, and pGL4.13 encoding luciferase were propagated in a DH5-strain of E. coli and purified by using a QIAGEN Plasmid Maxi Kit (Germany). YOYO-1, LysoTracker Red DND-99, DIO, dextran rhodamine B (MW 70 kDa) were purchased from Invitrogen (USA). The 5-TAMRA-SE was purchased from MedChem Express (USA). Fetal bovine serum (FBS) and RPMI 1640 medium were purchased from Thermo Fisher Scientific (USA). Hoechst 33342 Staining Solution for Live Cells, DAPI staining solution, RIPA lysis buffer, 0.25% trypsin-EDTA, BCA microplate protein assay kit, and ROS detection kit were obtained from Beyotime Institute of Biotechnology (Haimen, China). Luciferase Assay System Kit was purchased from Promega (USA). Anti-DR5, anti- TRAIL, and anti-GAPDH were purchased from Cell Signaling Technology (USA). All of the other reagents were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Branch PEI25K was purchased from Sigma-Aldrich (USA).
3
Cell Cycle Analysis and Apoptosis Quantification
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The cells were digested by 0.25% trypsin-EDTA (Beyotime Institute of Biotechnology, Haimen, China) and collected by centrifugation at 500 × g at 4°C for 5 min and washed three times with phosphate-buffered saline (PBS). The precipitated cells were resuspended and fixed in 70% absolute alcohol. Subsequently, for the purpose of analyzing cell cycle, the cells were washed with PBS and centrifuged at 500 × g at 4°C for 5 min to collect the precipitate. Propidium iodide (PI) was added for staining. The cell cycle status was determined using an EPICS XL-MCL FCM system (Beckman Coulter, Inc., Brea, CA, USA).
Cells in the logarithmic phase were collected and seeded into 6-well plates (3×105/well). The cells were then digested in EDTA-free trypsin (Shanghai Lanpai Biotechnology Co., Ltd., Shanghai, China), and stained with Annexin V-FITC and PI (Shanghai Lanpai Biotechnology Co., Ltd.). The cells were then incubated in the dark for 15 min at room temperature. The apoptotic rate of the cells in each group was detected using the EPICS XL-MCL FCM (Beckman Coulter, Inc.) with an excitation wavelength of 488 nm and emission wavelength of 530 nm.
Cells in the logarithmic phase were collected and seeded into 6-well plates (3×105/well). The cells were then digested in EDTA-free trypsin (Shanghai Lanpai Biotechnology Co., Ltd., Shanghai, China), and stained with Annexin V-FITC and PI (Shanghai Lanpai Biotechnology Co., Ltd.). The cells were then incubated in the dark for 15 min at room temperature. The apoptotic rate of the cells in each group was detected using the EPICS XL-MCL FCM (Beckman Coulter, Inc.) with an excitation wavelength of 488 nm and emission wavelength of 530 nm.
4
Synthesis of Multifunctional Nanoparticles
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Gold chloride trihydrate (HAuCl4·3H2O, 99.9%), sodium borohydride (NaBH4, 98%), trisodium citrate (99.5%), gadolinium chloride (GdCl3, 99%), hydrochloric acid (HCl, 36.0–38.0%), nitric acid (HNO3, 65.0–68.0%), silver nitrate (AgNO3, 99.8%), l -ascorbic acid (99.7%), sodium hydroxide (NaOH, 96.0%), cyclohexane (99.7%), tetraethyl orthosilicate and ethanol absolute (C2H6O, 99.7%) were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Triethanolamine (TEA, AR), bromoacetic acid (AR), hexadecyltrimethylammonium bromide (CTAB, 99%) and cetyltrimethylammonium (CTAC, 97%) were bought from Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China). 3-(Trimethoxysilylpropyl)diethylene triamine was purchased from Gelest (Morrisville, US). Fetal bovine serum (FBS, Gibco), phosphate buffered saline (PBS), trypsin–EDTA and Dulbecco's modified Eagle medium (DMEM) were obtained from Beyotime Biotechnology (Nantong, China).
5
Mouse Preosteoblastic MC3T3-E1 Cell Culture
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The mouse preosteoblastic MC3T3-E1 cell line was purchased from the Chinese Academy of Sciences Cell Bank. The cells were cultured in MEM-ALPHA medium (Biological Industries; cat. no. 01-042-1A) with 10% FBS (Biological Industries; cat. no. 04-001-1A) and 1% penicillin-streptomycin at 37°C, in the presence of 5% CO2 in a humidified incubator. The substrate was cultured in an incubator containing 5% CO2 at 37°C. When the cells reached 80–90% confluence, 0.25% trypsin-EDTA (Beyotime Institute of Biotechnology; cat. no. C0201) was used for digestion and subculture. The cells in the logarithmic growth stage were selected for the following experiments.
6
Isolation and Culture of Rabbit Amniotic Fluid-Derived Mesenchymal Stem Cells
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AFMSCs were isolated from rabbit amniotic fluid, as previously described (17 (link)). AFMSCs were cultured on a 100 mm diameter-plate (37°C, 5% CO2) in a growth medium consisting of low glucose Dulbecco's modified Eagle's medium (L-DMEM; Beyotime Institute of Biotechnology, Shanghai, China) supplemented with 20% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin and streptomycin (Beyotime Institute of Biotechnology), and detached from the substrate by incubation in 0.25% trypsin/EDTA (Beyotime Institute of Biotechnology). The cells were subsequently reseeded at a split ratio of 1:3 when growing to 90% confluence in culture.
7
Nanogel Synthesis and Characterization
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GOx nanogel (GN) and GOx-CAT nanogel (GCN) were synthetized in our previous study(Luo et al., 2021b (link); Fan et al., 2022 (link)). Trypsin-EDTA (ethylenediaminetetraacetic acid) was obtained from Beyotime Biotechnology. Dimethyl sulfoxide (DMSO) was supplied by Sinopharm Chemical Reagent Co. Ltd. Phosphate buffered saline (PBS) and bovine serum albumin (BSA) were supplied by Solarbio Technology Co., Ltd. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), penicillin-streptomycin, hematoxylin eosin (H&E staining kit), calcein-AM/PI double stain kit, and antifade mounting medium with DAPI (4ʹ,6-diamidino-2-phenylindole) were obtained from Yeasen Biotechnology Co. Ltd. Anti-fluorescence quenching tablet seal (including DAPI) was obtained from Shandong Sparkjade Biotechnology Co., Ltd. Fetal bovine serum (FBS) was obtained from Thermo Fisher Scientific Co., Ltd. A blood glucose meter was supplied by Sinocare Inc. A JPB-607A portable dissolved oxygen tester and a PHS-3C pH meter were supplied by Shanghai INESA Scientific Instrument Co., Ltd. The instruments used in the experiments were all from the Core Facility of Biomedical Sciences, Xiamen University.
8
Isolation and Culture of Human Bone Marrow Mesenchymal Stem Cells
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This study was approved by the Ethical Committee of Tongji Medical College, Huazhong University of Science and Technology. Human bone marrow aspirates were collected from healthy donors with informed consent. Low-density mononuclear cells (MNCs) were separated by Ficoll gradient centrifugation (Haoyang Biological, Tianjin, China) and cultured in Dulbecco's modified Eagle's medium (DMEM; Hyclone, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) and 1% penicillin–streptomycin (Beyotime, Shanghai, China) in a humidified incubator under an atmosphere of 5% CO2/95% air at 37°C. Nonadherent cells were removed by replacing the medium after 48 h of incubation. Cell passaging was performed when the monolayer of adherent cells reached 80% confluence with 0.25% trypsin-EDTA (Beyotime).
9
Isolation and IL-1β Stimulation of Chondrocytes from OA Cartilage
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As previously described (9 (link)), cartilage tissues from the knee joints of OA patients were collected, and then cut into small sections. After washing five times with PBS (Beyotime Institute of Biotechnology), the specimens were digested in 0.25% trypsin-EDTA (Beyotime Institute of Biotechnology) solution for 30 min and supplemented with Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc.) containing collagenase type II for 2 h at 37˚C. The released chondrocytes were seeded in 25 cm2 cell flasks. The cells were passaged at a ratio of 1:3, when they reached 80-90% confluency. For stimulation with IL-1β, the isolated chondrocytes were maintained in DMEM (Thermo Fisher Scientific, Inc.) at 37˚C, 5% CO2 and incubated with IL-1β (10 ng/ml; Abcam) for 24 h.
10
Glioma Cell Culture Protocol
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Penicillin, streptomycin, trypsin-EDTA (ethylene diaminetetra acetic acid) were purchased from Beyotime (Beijing, China). The human glioma cell lines U251, U343, U87, T98G, and Hs683 were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in and cultured in DMEM (Invitrogen, Shanghai, China), containing 10% fetal bovine serum (Gibco, Logan, UT, USA) and 100 U Penicillin and streptomycin at 37 °C in a humidified atmosphere containing 5% CO2.
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