Cells, analyzed for total protein expression, were washed with 1× phosphate-buffered saline (PBS), trypsinized with 500 μl of trypsin-EDTA (BioConcept) for 5 min, resuspended in 500 μl of ice-cold DMEM supplemented with 10% FBS and 1% PenStrep, and transferred to a microcentrifuge tube. The cells were pelleted by centrifugation for 20 min at 1000 rpm at 4°C, and the supernatant was discarded. The pellet was resuspended in 150 mM NaCl, 50 mM tris-HCl (pH 8.0) supplemented with deoxyribonuclease I. The cells were sonicated for five cycles as described in protein purification, and the cells were diluted in 4× SDS–polyacrylamide gel electrophoresis loading buffer.
Trypsin edta
Manufactured by BioConcept
Sourced in Switzerland
Trypsin-EDTA is a cell dissociation reagent used to detach adherent cells from cell culture surfaces. It contains the enzyme trypsin and the chelating agent EDTA, which work together to break down the extracellular matrix proteins and cell-cell adhesions, allowing cells to be harvested and subcultured. The product is commonly used in cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Collagenase type 1, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by BioConcept (2 mentions)
Penicillin g, by Thermo Fisher Scientific (1 mentions)
3 3 dihexyloxacarbocyanine iodide dioc6 3, by Thermo Fisher Scientific (1 mentions)
2 7 dichlorodihydrofluorescein diacetate h2dcfda, by Thermo Fisher Scientific (1 mentions)
Caspase 3, by R&D Systems (1 mentions)
Pan caspase inhibitor z vad fmk, by Merck Group (1 mentions)
Thiazolyl blue tetrazolium bromide mtt, by Merck Group (1 mentions)
Cisplatin, by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by BioConcept (1 mentions)
Fetal calf serum (fcs), by BioConcept (1 mentions)
Arpe 19, by American Type Culture Collection (1 mentions)
Foetal bovine serum (fbs), by PAN Biotech (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
5 protocols using trypsin edta
1
Protein Expression and Extraction
2
Apoptosis Induction in Cancer Cells
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Cisplatin, propidium iodide (PI) and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Trypsin-EDTA was purchased from BioConcept (Allschwil/BL, Switzerland). Fetal bovine serum (FBS), L-glutamine, penicillin G, 2’, 7’-dichlorodihydrofluorescein diacetate (H2DCFDA) and 3, 3-dihexyloxa-carbocyanine iodide [DiOC6(3)] were obtained from Thermo Fisher Scientific (Carlsbad, CA, USA). Caspase-3 and caspase-9 activity assay kits were purchased from R&D Systems Inc. (Minneapolis, MN, USA). The primary antibodies against Bcl-2, Bax, cytochrome c, Apaf-1, AIF, p21, cyclin A, cyclin D, cyclin E, CDK 2, β-actin and the goat anti-rabbit or anti-mouse IgG-horseradish peroxidase (HRP) secondary antibodies were purchased from GeneTex, (Hsinchu, Taiwan). Pan-caspase inhibitor (z-VAD-fmk) and enhanced chemiluminescence (ECL) detection kit (Immobilon Western Chemiluminescent HRP Substrate) were purchased from Merck Millipore (Billerica, MA, USA). YC-1 was designed and synthesized as detailed in the previous study [21 (link)].
3
Cell Culture and Characterization of Human Osteosarcoma and Retinal Pigment Epithelial Cell Lines
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The human osteosarcoma cell line MG-63 (CRL-1427) and human retinal pigment epithelial cell line ARPE-19 (CRL-2302), which were used as control, were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cell lines used were accompanied by identification test certificates and were grown according to corresponding tissue culture collection protocols. The ARPE-19 cells were grown in Dulbecco’s minimal essential medium (DMEM)/F12 and MG-63 were grown in DMEM supplemented with 10% Fetal calf serum (FCS) and 1% Penicillin-Streptomycin Solution at 37 °C, 5% CO2 and 100% humidity. The FCS, DMEM and Trypsin EDTA solution were from Bioconcept (Allschwil, Switzerland). All other chemicals employed in this study were from Merck (Buchs, Switzerland) and of the highest grade of purity. All the cell culture experiments were performed in TPP plastic ware (Trasadingen, Switzerland).
4
Isolation and Expansion of Rat Adipose-Derived Stem Cells
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All the in vitro studies were conducted in accordance with the local veterinary commission in Basel, Switzerland (No. 2925). Visceral adipose tissue was harvested from adult Sprague-Dawley rats and processed under sterile conditions as described earlier [21 (link)]. Briefly, the fat tissue was rinsed in 0.01 M phosphate-buffered solution (PBS), minced and resulting tissue was digested with 0.15% (w/v) Type I Collagenase (Gibco Life Technologies, Cat. No. 17100017) for 1 h at 37 °C and centrifuged for 5 min at 1500 rpm and 4 °C. The pellet was re-suspended in growth medium (GM) i.e., Dulbecco`s Modified Eagle`s Medium (DMEM, Gibco, Cat. No. 41965039) supplemented with 10% Foetal Bovine Serum (PAN-Biotech, EU-approved, Cat. No. P40-47500) and 1% Penicillin/Streptomycin (BioConcept, Cat. No. 4-01F00-H). Isolated ASC were seeded at a density of 3000 cells/cm2 and expanded at 37 °C with 5% CO2 in a humid atmosphere; GM was changed every 72 h. Cells were passaged using 0.25% Trypsin-EDTA (BioConcept, Cat. No. 5-51F00-H) at 90% confluence and resulting cells at passage 2 (P2) or 3 (P3) were used for the experiments.
5
Isolation and Culture of Adipose-Derived Stem Cells
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Adipose tissue was obtained from healthy patients undergoing elective liposuction or abdominoplasty. All patients had signed an informed consent form prior to the surgery. The isolation of ASC was performed under sterile conditions according to a well-established protocol [39 (link)]. The fat tissue was cleaned from erythrocytes by rinsing with 0.01M phosphate-buffered solution (PBS) and centrifugation, then minced and digested with 0.1% (w/v) Type I Collagenase (Gibco Life Technologies, Basel, Switzerland, Cat. No. 17100017) for 3 h at 37 °C and centrifuged for 5 min at 1500 rpm and 4 °C. The resulting pellet was resuspended in growth medium (GM), i.e., Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, Cat. No. 41965039) supplemented with 10% fetal bovine serum (FBS) (PAN-Biotech, Aidenbach, Germany, EU-approved, Cat. No. P40-47500) and 1% penicillin/streptomycin (BioConcept, Allschwil, Switzerland, Cat. No. 4-01F00-H). Extracted ASC were seeded at a density of 3000 cells/cm2 and cultured at 37 °C with 5% CO2 in a humid atmosphere, with GM being changed every 72 h. Cell passage was performed at 90% confluence by using 0.25% trypsin—EDTA (BioConcept, Cat. No. 5-51F00-H) and resulting cells at passage 2 (P2) or 3 (P3) were used for the experiments.
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