The cytotoxicity of the Fe3O4 and Co@Fe3O4 nanozymes with the addition of 10 nM H2O2 was determined using the CCK-8 cell viability assay kit (Dojindo Molecular Technologies). Briefly, A-498 cells (Human renal cancer cell, ATCC, HTB-44) were plated in 96-well plates (BD Biosciences) with the density of 5 × 103 cells per well and cultured in 100 μL EMEM (Catalog No. 30-2003) for 1 day before the addition of Fe3O4,Co@Fe3O4 nanozymes, or only the buffer as a control. On each plate, blank wells (n = 6) with media were defined as 0% viability. Moreover, the wells with only PBS-treated cells (n = 6) were defined as 100% viability. The dilutions of the Fe3O4 and Co@Fe3O4 nanozymes were prepared using a buffer containing 10 nM H2O2. The cells were then exposed to the Fe3O4 or Co@Fe3O4 nanozymes at a series of concentrations (from 0 to 0.2 mg mL−1) for 24 hours. After stimulation, a 10 μL CCK-8 solution was added to each well. The plates were then incubated for 4 h at 37 °C. After this, the absorbance was determined at 450 nm using the Benchmark Plus microplate spectrophotometer (Bio-Rad Laboratories, Inc.). The results presented herein are the average of those obtained via three independent experiments.
Cck 8 cell viability assay kit
Manufactured by Dojindo Laboratories
Sourced in Japan
The CCK-8 (Cell Counting Kit-8) is a colorimetric assay kit for measuring the viability and proliferation of cells. The kit utilizes a water-soluble tetrazolium salt that is reduced by dehydrogenase enzymes in living cells, producing a colored formazan dye that can be measured spectrophotometrically.
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Lab products found in correlation
Benchmark plus microplate spectrophotometer, by Bio-Rad (1 mentions)
96 well plate, by BD (1 mentions)
Enspire multimode plate reader, by PerkinElmer (1 mentions)
Elx800, by Agilent Technologies (1 mentions)
Gsh glo kit, by Promega (1 mentions)
Cobalt 2 chloride hexahydrate cocl2.6h2o, by Merck Group (1 mentions)
Gapdh antibody, by Santa Cruz Biotechnology (1 mentions)
Polyvinylpyrrolidone, by Merck Group (1 mentions)
Polyacrylic acid, by Merck Group (1 mentions)
Modular incubator chamber, by Biospherix (1 mentions)
Lactate dehydrogenase ldh release, by Beyotime (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
δψm assay kit with jc 1, by Beyotime (1 mentions)
8 protocols using cck 8 cell viability assay kit
1
Cytotoxicity Evaluation of Nanozymes
2
Cell Viability Assay for CPPs
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The viability of cells treated with CPPs was determined by the CCK-8 cell viability assay kit (Dojindo, Japan). Cells were seeded at a density of 1x106 cells/ml in a 96-well plate and incubated at 37°C. After indicated concentrations of CPPs treatment for 24 h, the medium was replaced with 110 μL of the fresh medium, containing 10% CCK8 reagent. After half an hour, the optical density was measured using micro-plate reader at 450 nm.
3
E2 Toxicity on Bone Marrow Stromal Cells
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The toxicity of E2 on BMSCs was measured by the CCK-8 assay. BMSCs were seeded in a 96-well plate and then cultured in CM with different doses of E2 (0 nM, 1 nM, 10 nM, 100 nM, 500 nM, and 1 μM) for 24 h and 48 h. The CCK-8 assays were performed using the CCK-8 Cell Viability Assay Kit (Dojindo, Cat. No. CK04-01, Kumamoto) according to the manufacturers' instruction.
4
Cytotoxicity Assessment of JPBuFr on C2C12 Myotubes
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The cytotoxicity of JPBuFr on C2C12 myotubes was evaluated using a cell counting kit-8 (CCK-8) cell viability assay kit (Dojindo, Kumamoto, Japan) according to the manufacturer's instructions. Briefly, differentiated C2C12 myotube cells (1 × 104 cells/well) were cultured onto 48-well plates and then incubated with JPBuFr (5, 10, and 20 μg/ml) alone or co-treated with JPBuFr (5, 10, and 20 μg/ml) and Dex (5 μM) for 24 h at 37 °C in a humidified atmosphere of a 5% CO2 incubator. Subsequently, 20 μL of 2-(2-Methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium Sodium Salt (WST-8) solution was added to each well followed by a 4 h incubation. The absorbance was measured using an EnSpire Multimode Plate Reader at 450 nm (PerkinElmer, MA, USA).
5
Cell Proliferation Measurement using CCK-8 Assay
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A cell counting kit‐8 (CCK‐8) cell viability assay kit (Dojindo Laboratories, Kumamoto, Japan) was used to determine the cell proliferation. In brief, cells were incubated in a 96‐well plate with 5 × 103 cells/well for 48 h. Then, we added 10 μL of cell viability assay kit solution to each well. After 2 h incubation at room temperature without light, the absorbance of each well was measured at 450 nm using a microplate reader (ELx800; Biotek, Winooski, VT, USA).
6
Cobalt-Induced Oxidative Stress Mitigation
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Polyacrylic acid (PAA, MW 1,800), polyvinylpyrrolidone (PVP, MW 29,000), potassium permanganate (KMnO4), L-glutathione reduced (GSH), manganese chloride tetrahydrate (MnCl2·4H2O), 2',7'-dichlorofluorescein diacetate (DCF-DA), and cobalt (II) chloride hexahydrate (CoCl2·6H2O) were purchased from Sigma-Aldrich (St. Louis, Mo, USA). A thiol detection assay kit was purchased from Abnova (Taipei City, Taiwan). Biotech cellulose ester dialysis membrane (Spectra/Por®, molecular weight cut off 100 kDa) was obtained from Spectrum Labs (Laguna Hills, CA, USA). A CCK-8 cell viability assay kit was obtained from Dojindo (Kumamoto, Japan). A GSH-Glo kit was purchased from Promega (Madison, WI, USA). Cyclin B1 and cyclin D1 antibodies were purchased from Cell signaling Technology (Danvers, MA, USA). GAPDH antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). All chemicals and solvents were used without further purification.
7
Assessing Hypoxia/Reoxygenation Injury in Primary Hepatocytes
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Primary hepatocytes were isolated as we previously described [24 (link)]. For hypoxia/reoxygenation assays, hepatocytes were cultured at 37°C in a modular incubator chamber (BioSpherix, Lacona, NY, USA) gassed with a 5% CO2 and 95% N2 gas mixture for 4 h followed by 2 h reoxygenation under normoxic conditions (air/5% CO2). Alda-1 (20 μM), 3MA (5 mM), or CC (5 μM) was added to the medium for 1 hour before hypoxia. Cell viability was assessed using the CCK8 Cell Viability Assay Kit (Dojindo, Japan), and cytotoxicity was determined by lactate dehydrogenase (LDH) release (Beyotime, Shanghai, China) according to the manufacturer's instructions. All experiments were performed at least 3 times to confirm the results. Mitochondrial transmembrane potential in hepatocytes was assessed by the ΔΨm assay kit with JC-1 (Beyotime) according to the manufacturer's instructions. Mitochondrial generation of superoxide was stained with MitoSOX Red (Invitrogen, Waltham, MA, USA).
8
CCK-8 Assay for Cell Viability
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CCK-8 cell viability assay kit (Dojindo Molecular Technologies, Inc., Tokyo, Japan) was used to detect cell viability rendering to the protocol of the manufacturer. Briefly, 1 × 105 cells/mL in a volume of 100 µL DMEM in 96-well plates were hatched for 24 h and then cured with DON for 24 h. After 3 h incubation with 10 µL CCK-8 reagent in each well, absorption at 450 nm was determined on a plate photometer (Thermo Scientific, Waltham, MA, USA).
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