Human normal ureteral tissues came from patients undergoing radical nephrectomy. After collected, ureteral tissues were washed thrice with PBS at room temperature, and then split open with eye scissors, and washed with PBS three times again. Then, the mucosa was separated from the ureteral tissues by forcep and minced into 1 mm pieces. These pieces were gathered and placed onto a 6-cm petri dish and treated with 0.05% EDTA-trypsin (Solarbio) for 25 min at room temperature. The digestion carried out by trypsin was terminated with α-MEM containing 10% fetal bovine serum, and centrifuged for 5 min at 1590 × g. Then the suspension was discarded and the pellet was resuspended with 1.5 mL of keratinocyte serum-free media (KSFM; Gibco, USA) and added onto a 10-cm petri dish gently, then cultured in the incubator at 37°C in a humidified atmosphere of 5% CO2. After 48 h of incubation, the outgrowth of cells from each piece of ureteral tissue can be observed, and 3 mL of additional KSFM was added to support cell growth. When UCs were 95% confluent, they were passaged by 0.05% EDTA-trypsin (Solarbio), and passages 3 to 6 of UCs were used to conduct further experiments.
Trypsin edta
Manufactured by Solarbio
Sourced in China
Trypsin-EDTA is a laboratory reagent used to dissociate adherent cells from cell culture surfaces. It contains the proteolytic enzyme trypsin and the chelating agent EDTA, which work together to break down the cell-to-cell and cell-to-substrate adhesions, allowing the cells to be suspended for further experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Solarbio (17 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (12 mentions)
Phosphate buffered saline (pbs), by Solarbio (5 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (5 mentions)
Fetal bovine serum (fbs), by Solarbio (5 mentions)
Streptomycin, by Solarbio (4 mentions)
Rpmi 1640 medium, by Solarbio (4 mentions)
Penicillin, by Solarbio (3 mentions)
Penicillin streptomycin, by Merck Group (3 mentions)
Dimethyl sulfoxide (dmso), by Solarbio (3 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Microscopy digital camera system, by Olympus (2 mentions)
Histostain plus kit, by ZSGB-BIO (2 mentions)
Il 1β, by Cell Signaling Technology (2 mentions)
Goat anti rabbit secondary antibody, by ZSGB-BIO (2 mentions)
Fetal bovine serum (fbs), by ExCell Bio (2 mentions)
Crystal violet, by Solarbio (2 mentions)
Rpmi 1640, by Solarbio (2 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Solarbio (2 mentions)
65 protocols using trypsin edta
1
Isolation and Culture of Human Ureteral Cells
2
Zey Modulates MMP2/9 Expression
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Cells were inoculated in 6-well plates (2.5×104 cell/well) and cultured in an incubator for 24 h. Cells were treated with different concentrations of Zey for 48 h at 10 (Zey1), 20 (Zey2) and 40 (Zey3) µmol/l. Untreated cells served as a control. Subsequently, cells were digested with 0.25% EDTA-trypsin (Beijing Solarbio Science & Technology Co., Ltd.) and centrifuged at 800 × g for 5 min at 4°C. Following centrifugation, cells were resuspended in F-12K medium. The expression of MMP2/9 was detected using ELISA test kits (cat. nos. MMP200 and DMP900, respectively; R&D Systems, Inc., Minneapolis, MN, USA). The standard curve was plotted with standard samples. The assay diluent (50 µl) was added to each well. In total, 50 µl sample in each group was added into each well. The plates was covered with plate sealer and maintained for 2 h at room temperature. Following washing, the conjugate reagent was added into each well and the plate was placed on a shaker at room temperature for 2 h. The substrate solution was added into each well and maintained for 30 min at room temperature. Subsequently, the stop solution was used to terminate the reaction. Finally, the absorbance at 450 nm was read using a FilterMax F3/F5 microplate reader.
3
Isolation and Culture of Human Adipose-Derived Stem Cells
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Human adipose tissue samples were obtained from the abdomen of healthy females, and informed consent was approved by the institutional review boards. The collected samples were sliced, treated with 0.1% collagenase type I (Sigma-Aldrich, USA) under agitation for 90 min at 37°C, filtered through a 100 μm mesh, and centrifuged at 3,000 × g for 10 min. The supernatant was discarded, and the precipitate was resuspended with α-MEM (Corning) containing 10% fetal bovine serum (FBS, Corning) and 100 U/ml penicillin/streptomycin (Keygen, China). Finally, the cell suspension was seeded onto the 10 cm Petri dishes, then maintained over 4–5 days until confluence was reached, which was defined as passage 0. When cells reached 80–90% confluence, they were passaged by 0.25% EDTA-trypsin (Solarbio, China).
4
Cell Cycle and Apoptosis Analysis
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Cell samples were treated with 0.25% EDTA–trypsin (Solarbio Life Sciences, Beijing, China) and centrifuged at 1000 rpm for 10 min at 4 °C, then the supernatant was removed, washed with PBS that had been precooled at 4 °C, and centrifuged again for 10 min. The cell cycle was detected via flow cytometry using the Cell Cycle and Apoptosis Analysis Kit (Yeasen, Shanghai, China). Cell apoptosis was detected via flow cytometry (AccuriC6, BD Biosciences, San Jose, CA, USA.) using the Annexin V-FITC/PI Apoptosis Detection Kit (Yeasen, Shanghai, China).
5
Quantification of NEFA in Cells
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The content of NEFA was measured using a NEFA Detection kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan), following the manufacturer's protocol. Briefly, cells were treated with drugs and grouped as aforementioned. Following treatments, 22 µM [9,10-3H]oleate was added to cells for 4 h. Cells were subsequently washed two times with PBS and cultured in DMEM for 4 h. The cells were digested with 0.25% EDTA-trypsin (Beijing Solarbio Science & Technology Co., Ltd.), resuspended in DMEM and centrifuged with 1,000 × g for 10 min. The supernatant was transferred into a clean centrifuge tube. Lipid was extracted from supernatant using N-hexane: Isopropanol solution (3:2) and a thin layer chromatography silica gel plate (25 (link)). The silica gel of containing NEFA fraction was scraped from the silica gel plate and dissolved in N-hexane: Isopropanol solution (3:2). Scintillation solution was added and the 3H-labeled NEFA protein content in the cells was measured using a L6500 liquid scintillation counter (Beckman Coulter, Inc., Brea, CA, USA). NEFA protein concentration was detected using a Bradford protein assay kit (Beyotime Institute of Biotechnology, Haimen, China).
6
Antimicrobial and Antioxidant Assays
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α-Glucosidase, α-amylase, and acarbose, 2,2-diphenyl-1-picrylhydrazyl (DPPH), were from Sigma-Aldrich (St. Louis, MO, USA), naphthyl ethylenediamine dihydrochloride, gallic acid, quercetin, sodium nitrate, methanol, trisaminomethane (Tris), nitro blue tetrazolium chloride (NBT), 1,4 Dihydronicotinamide adenine dinucleotide (NADH), phenazine methosulfate (PMS), FeSO4, saffron, salicylic acid, sodium nitroprusside, sulfanilic acid reagent, glacial acetic acid, and naphthyl ethylene diamine dihydrochloride were acquired from Sigma-Aldrich (Shanghai, China), 3-(4,5-dimethylthaizol-2-yl)-2,5-diphenytetrazolium bromide (MTT) was procured from Enzo Life Science (Plymouth Meeting, PA, USA). D-Hank’s buffer, dimethyl sulfoxide (cell-culture grade), and 0.25% EDTA trypsin were obtained from (Solarbio, China); RPMI 1640 medium and fetal bovine serum were acquired from (GIBCO, USA). Microbial strains; Escherichia Coli ATCC 8739, Staphylococcus aureus ATCC 25923, Bacillus cereus ATCC 10876, and Salmonella enterica ATCC 14028 were obtained from Microbiologic (St. Cloud, Minnesota, USA).
7
Isolation and Culture of hBMSCs
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Based on previously described methods, hBMSCs were isolated from the bone marrow tissue and cultured (29 (link)). Briefly, the bone marrow was mixed with an equal volume of phosphate-buffered saline (PBS), and then spread on lymphocyte separation medium (LSM; Beijing Solarbio Science & Technology Co., Ltd.; 1:1, v/v). After washing with PBS, the mononuclear cell layer was collected following centrifugation at 500 × g for 30 min at room temperature. The cells were then cultured in low-glucose DMEM (Beijing Solarbio Science & Technology Co., Ltd.) containing 10% (v/v) fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc.) and 100 units/ml penicillin-streptomycin (Beijing Solarbio Science & Technology Co., Ltd.), and maintained in a humidified incubator (HERAcell vios 160i, Thermo Fisher Scientific, Inc.) at 5% CO2 and 37°C. When 80-90% confluency was reached, the hBMSCs were detached using 0.05% trypsin-EDTA (Beijing Solarbio Science & Technology Co., Ltd.) and sub-cultured at a ratio of 1:2 in new culture flasks. The cells at passage 3 were used in subsequent experiments.
8
Flow Cytometric Analysis of Liver Organoids
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Liver organoids were treated with trypsin EDTA (0.25%; Solarbio) for 30 min and dissociated into single cells, which were then passed through 70-μm cell strainers. Subsequently, the HLO cells were fixed in 4% PFA for 20 min, permeabilized in 0.2% Triton X/PBS for 10 min, blocked with 1% BSA/PBS blocking solution for 30 min, and then incubated with primary antibodies for 1 h. Following incubation with secondary antibodies for 0.5 h in the dark, the stained cells were analyzed using BD FACS Celesta flow cytometer (BD Biosciences, Franklin Lakes, USA), and the obtained data were analyzed using FlowJo 10.0 software (Stanford University, Stanford, USA). A complete list of primary and secondary antibodies used is provided in Supplementary Table S2.
9
Cell Culture Protocol for L929 Cells
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Poly (tetrafluoroethylene) (PTFE) and gelatin were obtained from Sigma-Aldrich. Roswell Park Memorial Institute-1640 (RPMI-1640) was purchased from Gibco. Fetal bovine serum (FBS) was purchased from Shanghai Yeasen. DMSO, phosphate-buffered saline (PBS), penicillin-streptomycin (P/S), and trypsin-EDTA (0.025–0.01 wt%) were all purchased from Beijing Solarbio Science and Technology Co., Ltd. Live/dead kits were obtained from Invitrogen. The culture medium for L929 cells contained RPMI-1640 with 10% FBS and 1% PS. Milli-Q water (18.2 MΩ‧cm) was used in all experiments.
10
Fabrication and Characterization of Carboplatin-Loaded Nanoparticles
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Carboplatin (CBP) was purchased from Dalian Meilun Biotech Co., Ltd. (Dalian, China). Polylactic acid-Polyethylene glycol (PLA-PEG) was purchased from Shandong Academy of Pharmaceutical Science (Jinan, China). 3-(4, 5-Dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide (MTT) was purchased from Solarbio (Shanghai, China). 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR) was procured from Fanbo Biochemicals Co., Ltd (Beijing, China). Fluorescein isothiocyanate (FITC) was purchased from Sigma (MO, USA). DAPI was purchased from Sigma (USA). Doxorubicin hydrochloride (DOX•HCl) was purchased from Dalian Meilun Biotech Co., Ltd. (Dalian, China). Penicillin and streptomycin (1%), trypsin-EDTA (0.25%), phosphate and phosphate buffered saline (PBS) were the product of Solarbio Biotechnology Co. Ltd (Shanghai, China). Roswell Park Memorial Institute medium (RPMI-1640) were obtained from Hyclone. All materials and reagents used in this work were of analytical grade and used without further purification.
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