VK2/E6E7 were seeded in 96-well plates. Once 90–95% confluent were obtained, the cells were treated as described above and incubated at 37 °C under a humidified atmosphere containing 5% CO2 for 24 h. Then, 100 μL of MTT solution (5 mg/mL) (Servicebio Technology Co., China) was added to each well, and the cells were incubated at 37 °C under a humidified atmosphere containing 5% CO2 for 4 h. The supernatant was discarded and 100 μL of dimethyl sulfoxide (DMSO) (Servicebio Technology Co., China) was added to each well. Crystals were dissolved by shaking for 15 mins and absorbance was measured at 570 nm using a microplate reader (Bio-Tek, USA). Viability was calculated using the following formula: Cell survival rate = (model group OD value-blank pore OD value)/(normal control group OD value-blank pore OD value) × 100%.
Dimethyl sulfoxide (dmso)
Manufactured by Wuhan Servicebio Technology
Sourced in China
DMSO is a colorless, odorless, and aprotic solvent. It has a high boiling point and is miscible with water and many organic solvents. DMSO is commonly used as a cryoprotectant in cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Microplate reader, by Agilent Technologies (1 mentions)
Mtt solution, by Wuhan Servicebio Technology (1 mentions)
Deferoxamine mesylate, by Selleck Chemicals (1 mentions)
3 ma hy 19312, by MedChemExpress (1 mentions)
Z vad fmk s7023, by Selleck Chemicals (1 mentions)
Anti bcl 2, by Cell Signaling Technology (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
Anti phospho iκbα, by Cell Signaling Technology (1 mentions)
Anti bad, by Cell Signaling Technology (1 mentions)
Erastin hy 15763, by MedChemExpress (1 mentions)
Anti p65 sc 8008, by Santa Cruz Biotechnology (1 mentions)
Anti cyclin d1 60186 1 ig, by Proteintech (1 mentions)
Anti zeb 1 21544 1 ap, by Proteintech (1 mentions)
Anti nrf2 m200 3, by Medical & Biological Laboratories (1 mentions)
Anti gpx4 67763 1 ig, by Proteintech (1 mentions)
Anti gapdh 60004 1 ig, by Proteintech (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Cycloheximide (chx), by Selleck Chemicals (1 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (1 mentions)
Chloroquine cq, by MedChemExpress (1 mentions)
7 protocols using dimethyl sulfoxide (dmso)
1
MTT Cytotoxicity Assay Protocol
2
Ferroptosis Regulation by Molecular Inhibitors
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Myrislignan (S3261), Z-VAD-FMK (S7023), necrosulfonamide (S8251), ferrostatin-1 (S7243), deferoxamine mesylate (S5742), and RSL3 (S8155) were purchased from Selleck (USA). 3-MA (HY-19312) and erastin (HY-15763) were purchased from MedChemExpress (USA). Dimethyl sulfoxide (DMSO) was obtained from Servicebio (G5051, China). The following antibodies were used: anti-GAPDH (60004-1-Ig, Proteintech), anti-E-cadherin (14472, Cell Signaling Technology), anti-Snail-1 (13099-1-AP, Proteintech), anti-Slug (GTX128796, GeneTex), anti-p65 (sc-8008, Santa Cruz), anti-p-p65 (sc-136548, Santa Cruz), anti-phospho-IκB-α (2859, Cell Signaling Technology), anti-IκB-α (CSB-RA015761A0HU, Cusabio Technology), anti-SLC7A11 (NB300-318, Novus), anti-β-catenin (phospho Y142, ab27798, Abcam), anti-β-catenin (ab32572, Abcam), anti-Cyclin-D1 (60186-1-Ig, Proteintech), anti-Bad (9292, Cell Signaling Technology), anti-Bcl2 (15017, Cell Signaling Technology), anti-Vimentin (GB111308, Servicebio), anti-Twist (25465-1-AP, Proteintech), anti-ZEB1 (21544-1-AP, Proteintech), anti-Nrf2 (M200-3, Medical and Biological Laboratories), anti-TFR1 (ab214039, Abcam), and anti-GPX4 (67763-1-Ig, Proteintech).
3
Evaluating p53 Stability in GC Cell Line
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KGN, a human GC line, Obtained from ATCC, was cultured in DMEM/F12 medium (Gibco, Grand Island, NE, United States) supplemented with 10% fetal bovine serum (Gibco) and 100 μg/mL antibiotics (a mixture of penicillin and streptomycin; Welgene) at 37 °C in a 5% CO2 incubator.
The cells were treated with 10 μM of PFT-α (Selleck, USA) or equal dimethyl sulfoxide (DMSO; Servicebio, Wuhan, China) for 12 h and then the cells were digested with enzyme and collected for Western blotting. The cells were subjected to starvation for the indicated periods using EBSS (Procell, Wuhan, China) and detected via autophagy flux using 10 μM of chloroquine (CQ, MedChemExpress, Shanghai, China). Furthermore, the cells were treated with 50 of μg/mL cycloheximide (CHX, Selleck, State of Texas, USA) and collected every 30 min to determine the stability of the p53 protein.
The cells were treated with 10 μM of PFT-α (Selleck, USA) or equal dimethyl sulfoxide (DMSO; Servicebio, Wuhan, China) for 12 h and then the cells were digested with enzyme and collected for Western blotting. The cells were subjected to starvation for the indicated periods using EBSS (Procell, Wuhan, China) and detected via autophagy flux using 10 μM of chloroquine (CQ, MedChemExpress, Shanghai, China). Furthermore, the cells were treated with 50 of μg/mL cycloheximide (CHX, Selleck, State of Texas, USA) and collected every 30 min to determine the stability of the p53 protein.
4
Antibody-based Protein Expression Analysis
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The antibodies included the following: anti-SAA1 (YN3036, immunoway, USA), anti-GAPDH (#5174, Cell signaling Technology(CST), USA), anti-Bcl-2 (127891, Proteintech, USA), anti-Bax (50599-2, Proteintech), anti-Phospho-AKT(#4060, CST), anti-AKT (#4691, CST), anti-cleaved-caspase3 (ab32042, Abcam, UK), anti-caspase3 (19677-1-AP, Proteintech). AKT activator SC79 was purchased form Sellect (S786303, purity (>97%), USA) and dissolved in dimethyl sulfoxide (DMSO), which was obtained from Servicebio (G5051, Wuhan, China).
5
Autophagy Monitoring Using Lentiviral Vectors
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The recombinant lentivirus vector mRFP-GFP-LC3B was produced by GENECHEM (Shanghai, China). GFP and mRFP fluorescent puncta indicate autophagosomes that has not fused with lysosomes. However, when the autophagosome fuses with the lysosome, the GFP signal is sensitive to the acidic environment in the lysosome and is quenched; therefore, the mRFP punctum that do not emit GFP correspond to an autolysosome.
Rapamycin (Rapa), 3-methyladenine (3-MA) and bafilomycin A1 (Baf A1) were purchased from Selleck, and LY294002 was purchased from MCE (MedChemExpress, Shanghai, China). The following antibodies in the immunoblotting and immunofluorescence were used: anti-LC3B, anti-Atg5, anti-p-Akt, anti-Akt, anti-PI3K, anti-p-mTOR, anti-mTOR, anti-gD and anti-ICP5 were obtained from Cell Signaling Technology. The antibody for SQSTM1/p62 was obtained from Proteintech, and anti-β-actin, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium (MTT) and DMSO were purchased from Servicebio, Wuhan, China.
Rapamycin (Rapa), 3-methyladenine (3-MA) and bafilomycin A1 (Baf A1) were purchased from Selleck, and LY294002 was purchased from MCE (MedChemExpress, Shanghai, China). The following antibodies in the immunoblotting and immunofluorescence were used: anti-LC3B, anti-Atg5, anti-p-Akt, anti-Akt, anti-PI3K, anti-p-mTOR, anti-mTOR, anti-gD and anti-ICP5 were obtained from Cell Signaling Technology. The antibody for SQSTM1/p62 was obtained from Proteintech, and anti-β-actin, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium (MTT) and DMSO were purchased from Servicebio, Wuhan, China.
6
Evaluating p53 Inhibitor Effects on HVSMC Viability
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HVSMCs cultured for 3 days after conducted relevant genetic manipulation and pretreated with 10 μM of the specific p53 inhibitor PFT-α (Selleck, # S2929, USA) or equal dimethyl sulfoxide (DMSO; Servicebio, # WGD2650, China) for 12 h were seeded in 96-well plates at the density of 3 × 103/190 μl/well; each group had three repetitions. After continuing to treat with the same concentration of PFT-α or DMSO for the corresponding time, 10 μl of Cell Counting Kit-8 (CCK8) detection reagent (Beyotime Bio, #C0039, China) was added to each well and the optical density at 450 nm of each well was measured by microplate reader (Perkin Elmer, USA). The cell viability was determined every 12 hours for the next 3 days as described.
7
Comprehensive Biochemical Assay Protocol
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DMSO was purchased from Servicebio (GC203005). The AKT inhibitor SC66 was obtained from MedChemExpress (HY-19832). TMZ was purchased from Selleck (S1237).
The following antibodies were used: anti-RND1 (GTX83709, GeneTex), anti-GAPDH (#5174, Cell Signalling Technology, CST), anti-Flag (M185, Medical Biological Laboratories), anti-E-cadherin (20874–1-AP, Proteintech), anti-N-cadherin (22018–1-AP, Proteintech), anti-Vimentin (10366–1-AP, Proteintech), anti-Snail1 (sc -28,199, Santa Cruz), anti-mmp2 (10373–2-AP, Proteintech), anti-AKT (GTX121937, GeneTex), anti-Phospho-AKT (#4060, CST), anti-GSK-3β (#12456, CST), anti-Phospho-GSK-3β (#9323, CST), anti-β-catenin (51067–2-AP, Proteintech), anti-MAPK (#4511, CST), anti-Phospho-MAPK (#9212, CST) and anti-Smad3 (66516–1-Ig, Proteintech).
The following antibodies were used: anti-RND1 (GTX83709, GeneTex), anti-GAPDH (#5174, Cell Signalling Technology, CST), anti-Flag (M185, Medical Biological Laboratories), anti-E-cadherin (20874–1-AP, Proteintech), anti-N-cadherin (22018–1-AP, Proteintech), anti-Vimentin (10366–1-AP, Proteintech), anti-Snail1 (sc -28,199, Santa Cruz), anti-mmp2 (10373–2-AP, Proteintech), anti-AKT (GTX121937, GeneTex), anti-Phospho-AKT (#4060, CST), anti-GSK-3β (#12456, CST), anti-Phospho-GSK-3β (#9323, CST), anti-β-catenin (51067–2-AP, Proteintech), anti-MAPK (#4511, CST), anti-Phospho-MAPK (#9212, CST) and anti-Smad3 (66516–1-Ig, Proteintech).
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