Dimethyl sulfoxide was purchased from Sigma- Aldrich (Merck Millipore, Darmstadt, Germany). Dulbecco's modified Eagle's medium, fetal bovine serum (FBS), penicillin-streptomycin and trypsin-ethylenediaminetetraacetic acid were obtained from Welgene, Inc. (Gyeongsan-si, South Korea). Acridine orange (AO), ethidium bromide (EB) and 2′,7′-dichlorofluorescein diacetate (DCFH-DA) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Primers were obtained from Macrogen Inc. (Seoul, South Korea).
Trypsin ethylenediaminetetraacetic acid
Manufactured by Welgene
Trypsin-ethylenediaminetetraacetic acid (Trypsin-EDTA) is a commonly used laboratory reagent in cell culture applications. It is a mixture of trypsin, a proteolytic enzyme, and EDTA, a chelating agent. The primary function of Trypsin-EDTA is to facilitate the detachment of adherent cells from the culture surface by breaking down the cell-to-cell and cell-to-substrate adhesions. This solution is widely used in various cell culture protocols, such as cell passaging, cell harvesting, and cell dissociation.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Welgene (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Medium 199, by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Welgene (2 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (2 mentions)
Anti gapdh, by Abcam (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Cell lysis buffer, by Cell Signaling Technology (2 mentions)
Fetal bovine serum (fbs), by Welgene (1 mentions)
Dulbecco s modified eagle s medium, by Welgene (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Anti phospho enos ser1177, by Cell Signaling Technology (1 mentions)
Phenylmethylsulfonyl fluoride pmsf, by Merck Group (1 mentions)
Hepg2 cell, by American Type Culture Collection (1 mentions)
Endothelial cell medium (ecm), by ScienCell (1 mentions)
Lsecs, by ScienCell (1 mentions)
Sodium pyruvate, by Welgene (1 mentions)
Phosphate buffered saline pbs, by Biosesang (1 mentions)
9 protocols using trypsin ethylenediaminetetraacetic acid
1
Assessing Cellular Viability and Oxidative Stress
2
Isolation and Culture of Horse Muscle Cells
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The Pusan National University-Institutional Animal Care and Use Committee approved the study design (Approval Number: PNU-2015-0864). A skeletal muscle tissue biopsy was performed on the leg of a neonatal Thoroughbred to culture horse muscle cells (HMCs). HMCs were cultured according to the previous study [11 (link)]. Briefly, primary HMCs were maintained and sub-passaged in Medium 199 (Gibco, Grand Island, NY, USA) supplemented with 10% foetal bovine serum (Invitrogen, Carlsbad, CA, USA) and 1% antibiotic-antimycotic (Gibco, USA). HMCs were incubated in a humidified atmosphere with 5% CO2 at 37°C. Routine fluid renewals were made three times a week. At 70% to 80% confluence, cells were gently washed twice with phosphate-buffered saline and harvested using 0.05% trypsin-ethylenediaminetetraacetic acid (Welgene, Daegu, Korea) to isolate total RNA. We chose 42°C for heat stress according to the physiological condition of horse skeletal muscle tissue after exercise [12 (link)]. A value of 2% of O2 was determined to induce the hypoxic condition according to the previous work using a hypoxic chamber [13 (link)].
3
Heat Shock Response in Horse Muscle Cells
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Horse muscle cells were maintained and sub-passaged in DMEM (Gibco, Grand Island, NY, USA) and were supplemented with 10% foetal bovine serum (Invitrogen, Carlsbad, CA, USA) and 1% antibiotic-antimycotic (ABAM; Invitrogen, USA). The cells were cultured in a humidified atmosphere with 5% CO2 at 37°C. Routine fluid renewals were performed 3 times per week. At 70% to 80% confluence, cells were gently washed twice with phosphate-buffered saline and harvested using 0.05% trypsin-ethylenediaminetetraacetic acid (Welgene, Gyeongsan, Korea) for total RNA extraction. To apply heat shock, the 70% to 80% confluent horse muscle cells were transferred to an incubator at a temperature of 40°C for 1 h and 4 h followed by a 4 h recovery period in an atmosphere of 5% CO2.
4
Molecular Mechanisms in HepG2 Cell Culture
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HepG2 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). LSECs and endothelial cell medium were obtained from ScienCell Research Laboratory (Carlsbad, CA, USA). Phosphate-buffered saline, penicillin/streptomycin, and trypsin-ethylenediaminetetraacetic acid solutions were purchased from Welgene (Daegu, Korea). SP, phenylmethylsulfonyl fluoride (PMSF), and CDCA were provided by Sigma Aldrich (St. Louis, MO, USA). Anti-GAPDH, anti-ICAM-1 (EP1442Y), and anti-caspase-3 (E87) antibodies (Abcam, Cambridge, MA, USA) were also used. Anti-epithelial cadherin (E-cadherin) (24E10), anti-cleaved caspase-3 (Asp175), anti-endothelial nitric oxide synthase (eNOS), and anti-phospho-eNOS (Ser1177) antibodies and 10X cell lysis buffer were obtained from Cell Signaling Technology (Danvers, MA, USA).
5
Benzo[a]pyrene-Induced Cellular Responses
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Benzo[a]pyrene (B[a]P), myricetin, dimethyl sulfoxide (DMSO), ammonium persulfate, nuclease P1, N,N,N’N’-Tetramethyl ethylenediamine (TEMED), protease inhibitor cocktail, phosphatase inhibitor cocktail II, phosphatase inhibitor cocktail III, glycine, sodium dodecyl sulfate (SDS), tris base, sodium chloride, tween 20, and 2-mercaptoethanol were obtained from Sigma-Aldrich Chemical (St. Louis, MO, USA). Sodium pyruvate, penicillin-streptomycin, and trypsin-ethylenediaminetetraacetic acid (EDTA) were obtained from Welgene (Daegu, Korea). Alkaline phosphatase (AP) was purchased from Takara Bio Inc (Shiga, Japan). Phosphate-buffered saline (PBS) was purchased from Biosesang (Seongnam, Korea). A total of 30% acrylamide/bis solution was purchased from Bio-Rad (Hercules, CA, USA). Antibodies, such as CYP1A1, CYP1B1, GST, ABCC2, β-actin, and HRP-conjugated anti-rabbit immunoglobulin G (IgG), were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
6
Malonic Acid Modulates Oxidative Stress Response
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Malonic acid (MA), 2′, 7′-dichlorodihydrofluorescein diacetate (DCF-DA), dimethyl sulfoxide, and epigallocatechin-3-gallate (EGCG), as a positive control of ROS induction, were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), and trypsin-ethylenediaminetetraacetic acid were purchased from Welgene (Gyeongsan, Korea). SYBR green was purchased from GeneAll (Seoul, Korea). All antibodies (anti-heme oxygenase 1 (HO-1), anti-superoxide dismutase 1 (Sod1), anti-nuclear factor-erythroid 2-related factor-2 (Nrf2), anti-p-inhibitor of NF-κB (IκB), anti-IκB, anti-p-p65, anti-p65, anti-p50, anti-Cox-2, anti-IL-6, anti-TNF-α, anti-p-JNK, anti-JNK, anti-p-ERK, anti-ERK, anti-p-p38, anti-p38, anti-p-c-Jun, anti-c-Jun, anti-c-Fos, anti-p-Smad2/3, anti-Smad2/3, anti-collagen type I alpha 1 (Col1a1), anti-Col3a1, and anti-glyceraldehyde 3-phosphate dehydrogenase (Gapdh)) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
7
Oxidative Stress in Equine Myocytes
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A skeletal muscle tissue biopsy was performed on the leg of a neonatal Thoroughbred. Horse skeletal muscle cells were maintained and sub-passaged in Medium 199 (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA), 2% donor equine serum (DES; Hyclone, Carlsbad, CA, USA), and 1% antibiotic-antimycotic (ABAM; Invitrogen, USA). Medium 199 supplemented with 0.5% FBS and 1% ABAM was used as the differentiation medium. Horse skeletal muscle cells were cultured in a humidified atmosphere with 5% CO2 at 37°C. At approximately 70% to 80% confluence, cells were treated with 1 mM H2O2 (Junsei, Tokyo, Japan) and cultured for 6 h. Cells were gently washed twice with phosphate-buffered saline, and were then harvested using 0.05% trypsin-ethylenediaminetetraacetic acid (Welgene, Gyeongsan, Korea) to extract total RNA.
8
Isolation of Testicular Germ Cells
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Transportation of testes from a local farm (Gumbo Inc., Korea) to our laboratory was conducted in ice-cold Dulbecco’s phosphate-buffered saline (DPBS; Welgene Inc., Daegu, Korea) supplemented with 1% (v/v) antibiotic-antimycotic solution (Welgene, Korea) within 1 h. The tunica albuginea and epididymis of testis were removed and the seminiferous tubules were digested by 0.1% (w/v) type IV collagenase (Worthington Biochemical, Lakewood, NJ, USA) in high glucose Dulbecco’s modified Eagle’s medium (DMEM; Welgene, Korea) at 37°C for 15 min. Subsequently, the fragments of the seminiferous tubules were dissociated by 0.1% (w/v) hyaluronidase (Sigma-Aldrich, St. Louis, MO, USA) in high glucose DMEM at 37°C for 10 min and the dissociated fragment of the seminiferous tubule were promptly digested by 0.25% trypsin–ethylenediaminetetraacetic acid (Welgene, Korea) at 37°C for 10 min. After washing the dissociated testicular cells in DMEM supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; HyClone, Logan, UT, USA), they were filtered using a 70-μm nylon strainer (SPL, Pocheon, Korea) for eliminating myoid and Sertoli cells. Moreover, erythrocytes in the cells were removed by red blood cell lysis buffer (Sigma-Aldrich, USA) for 15 min at room temperature.
9
ARPE-19 Cell Culture and Analysis
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ARPE-19 cells were purchased from ATCC (Manassas, VA, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco (Grand Island, NY, USA). Phosphate-buffered saline, penicillin/streptomycin, and trypsin-ethylenediaminetetraacetic acid were purchased from Welgene (Daegu, Republic of Korea). SP and phenylmethylsulfonyl fluoride (PMSF) were obtained from Sigma–Aldrich (St. Louis, MO, USA). Anti-GAPDH, anti-ICAM-1, and anti-RPE65 antibodies (Abcam, Cambridge, MA, USA) were also used. Anti ZO-1, anti E-cadherin (24E10), anti-Akt, and anti-phospho-Akt (ser473) antibodies and cell lysis buffer were purchased from Cell Signaling Technology (Danvers, MA, USA).
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