The Cell Counting Kit-8 (APExBIO, Houston, Texas, USA) was used to examine the cellular viability. Briefly, Huh7 cells transfected with different plasmids (shNC, sh#1, sh#2, and sh#3) were seeded in 96-well plates at a density of 2×103 cells/well. At days 1–6, 10 µL CCK-8 reagent were mixed with 100 µL DMEM, and the mixed solution was added to each well and re-incubated for 2 h, followed by measurement of the absorbance at a wavelength of 450 nm using a microplate reader (Thermo MK3, Thermo Fisher Scientific, USA).
Thermo mk3
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Thermo MK3 is a versatile laboratory centrifuge designed for a wide range of applications. It features a robust and compact design, providing consistent and reliable performance. The Thermo MK3 can accommodate various rotor options to accommodate different sample volumes and types.
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Lab products found in correlation
Cell counting kit 8 (cck8), by Apexbio (2 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (1 mentions)
Streptavidin hrp antibody, by R&D Systems (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
Ifn γ, by Thermo Fisher Scientific (1 mentions)
Il 1α, by R&D Systems (1 mentions)
Cell growth determination kit, by Merck Group (1 mentions)
Prism 6, by GraphPad (1 mentions)
Cell counting kit 8 cck 8, by Apexbio (1 mentions)
Mrc complex activity assay kit, by Solarbio (1 mentions)
13 protocols using thermo mk3
1
Cellular Viability Assay with CCK-8
2
Cytotoxicity Assay of H2O2 in rMC-1 Cells
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rMC-1 cells were seeded onto 96-well plates and incubated at a density of 1 × 104 cells/well for 24 h. The cells were treated with hydrogen peroxide (H2O2; #323381, Sigma-Aldrich, St. Louis, MO, USA) at 0, 50, 100, 200, and 500 µM. The cell viability was detected with a Cell Counting Kit 8 (CCK8) (Dojindo, Japan); rMC-1 cells were treated with CCK8 at 37 °C for 1–4 h, and the absorbance at 450 nm was measured using a microplate reader (Thermo MK3, Thermo Fisher Scientific) to quantify the formazan products.
3
PACE4 Knockdown Impacts Cell Viability
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Cells were cultured in 96-well culture plates and transiently transfected PACE4 siRNA and control siRNA. After transfection, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to each well to a final concentration of 5 mg/mL in culture medium and incubated at 37°C for 4 hours. The reaction was terminated by removal of the supernatant and addition of 150 μL dimethyl sulfoxide (DMSO) to dissolve the formazan product. The plates were read at 405 nm on a microELISA plate reader (Thermo MK3; Thermo Fisher Scientific Inc). Each assay was performed at least three times.
4
Cytokine Quantification in Muscle Extracts
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96-well microplates were coated by 0.2 μg antibodies against IL-1α (R&D systems), IL-13 (R&D systems), IFN-γ (eBioscience) and TNF-α (eBioscience), respectively. Total proteins extracted from TA muscles were applied to the microplates. Biotinylated goat antibodies against IL-1α (R&D systems), IL-13 (R&D systems), IFN-γ (eBioscience) and TNF-α (eBioscience) were applied as detection antibodies, respectively. Streptavidin-HRP antibody (R&D systems) was used as the secondary antibody. The signals were collected by microplate reader (Thermo MK3, Thermo Fisher) and quantified by comparison with standard solution containing recombinant mouse IL-1α (R&D systems), IL-13 (R&D systems), IFN-γ (eBioscience) and TNF-α (eBioscience), respectively. The standard curves were made according to the manufacturer's instructions.
5
Evaluating Cytotoxicity of Drug Formulations
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The in vitro cytotoxicity of various formulations was studied by MTT assay.30 (link) Following attachment, cells were exposed to blank or DOX-loaded compounds (β-CD, mPEG5000-CD, PELA54, PELA54-CD) at a previously determined set concentration and incubated in thermostatic incubator for 48 hours. Then, 5 mg/mL MTT reagents were added into each well. After incubating for additional 4 hours in thermostatic incubator, the medium was discarded. Thereafter, 200 μL of DMSO was added to dissolve formazan. The plate was measured at 570 nm on a microplate reader (Thermo MK3; Thermo Fisher Scientific, Waltham, MA, USA). The inhibition rate of cell growth was calculated using the formula: Inhibitory rate (%) = (Acontrol − Asample)/Acontrol ×100. The cytotoxicity of various compounds was expressed as half maximal inhibitory concentration (IC50) values, which referred to the concentration that caused 50% inhibition of cell growth compared with untreated cells. The resistance reversion index (RRI) was defined as the ratio of IC50 of free DOX vs that of DOX-loaded micelles (RRI = IC50,free DOX/IC50,micelles).31 (link)
6
Sevoflurane-Induced Oxidative Stress Biomarkers
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Plasma and hippocampal homogenate were collected at 6 h following sevoflurane exposure. For plasma collection, blood samples were obtained from the left ventricle and transferred into heparinized tubes. The samples were centrifuged for 30 min at 3,000 × g at 4°C within 30 min of collection. Supernatants of the blood samples were stored at −80°C for later use in the plasma ELISA. For hippocampal homogenates, brain tissue was removed following rapid decapitation. Bilateral hippocampi were dissected in 0°C phosphate-buffered saline (PBS) on ice under an XTX-4A microscope (Xindiweiye Medical Instrument Co., Ltd., Wuhan, China). Hippocampal tissue was homogenized in 1 ml/g of 0°C PBS, and the homogenate was stored at −80°C for later use in ELISA. ELISA assays were performed according to the protocols of the commercial kits for rat superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione peroxidase (GSH-px; Kaysam Bio-Technology Co., Ltd, Guangzhou, China). The results were obtained by using a microplate reader (Thermo MK3, Thermo Fisher Scientific Inc., Waltham, MA, USA).
7
EGCG Cytotoxicity on HeLa Cells
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The viability of HeLa cells was measured using an MTT-based Cell Growth Determination kit (Sigma-Aldrich; Merck KGaA). Cells were seeded into 96-well flat-bottomed plates (10,000 cells in 100 µl/well), incubated overnight at 4°C and treated with different concentrations of EGCG (100, 80, 60, 40, 20 and 10 µg/ml) in triplicate for 0, 24 and 48 h, respectively. Cell culture medium with DMSO (final concentration, 0.1%; Invitrogen; Thermo Fisher Scientific, Inc.) was used as the negative controls. After drug treatment, 10 µl MTT solution was added to each well and the plates were incubated at 37°C for another 2 h. The supernatant was subsequently removed and DMSO was added to each well to dissolve the formazan. The absorbance/optical density (OD) was read at 492 nm using a Thermo MK3 spectrophotometric microplate reader (Thermo Fisher Scientific, Inc.). The inhibition rate of HeLa cells by EGCG was calculated using the following formula: Inhibition rate (%)=(1-ODdrug exposure/ODcontrol) ×100%. The half inhibition concentration (IC50) value of EGCG in inhibiting the growth of HeLa cells was measured by the MTT assay. Values were determined from the inhibition rate vs. drug concentration graphs using GraphPad Prism 6 software (GraphPad Software Inc., La Jolla, CA, USA).
8
Evaluating Cell Proliferation Assays
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Cell Counting Kit-8 (CCK-8), colony formation, and 5-Ethynyl-2′-deoxyuridine (EdU) were used for the cell proliferation assay. In brief, transfected cells were seeded in triplicate into 96-well plates (2000 cells per well) and cultured overnight in the same conditions. On days 1–5, CCK8 working solutions (Complete Media: CCK-8 = 10:1) were piped into the wells and incubated in the dark for 3 h. The absorbance (OD450) was detected using a microplate reader (Thermo MK3, Thermo Fisher Scientific, USA). For the colony formation assay, 1 × 103 cells were seeded in a 6-well plate and cultured for two weeks. Cells were fixed with 4% paraformaldehyde fixation and stained with 0.3% crystal violet. For the EdU (Cy3) assay, cells were incubated with Edu working solution (1:1000) for 2 h and then treated with paraformaldehyde for 20 min. After 3 washes in PBS supplemented with 3% BSA, the cells were permeabilized with 0.5% Triton for 15 min, followed by another 3 washes. After being incubated with the click reaction solution for 30 min in the dark, the cells were counterstained with Hoechst for 10 min. The images were captured using a fluorescence microscope (Olympus IX73, Japan).
9
Cell Viability Evaluation via Colony Assay and CCK-8
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Cell viability was assessed using the colony formation assay and the cell counting Kit-8 (CCK-8). For the colony formation assay, transfected cells were seeded into 6-well plates (1500 cells per well for HCCC-9810; 2000 cells per well for HUCCT1), gently shaken, and cultured at 37 °C for 14 days. The culture medium was replaced every 3 days. Cells were then fixed with 4% paraformaldehyde for 15 min, and stained with 0.1% crystal violet for 10 min. The number of colonies (diameter > 30 μm) was counted and plotted as the mean ± standard deviation of three independent experiments. For the CCK-8, 2 × 103 cells/well were seeded into 96-well plates. On days 1–5, the culture medium was replaced with the CCK-8 working solution (DMEM:CCK-8 = 100:1), and the plates were incubated in the dark for 2 h at 37 °C with 5% CO2. The absorbance (OD450) was finally measured using a microplate reader (Thermo MK3, Thermo Fisher Scientific, Waltham, USA).
10
Quantifying Cell Growth Capacity with CCK-8 Assay
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Cell growth capacity was assessed with the use of Cell Counting Kit-8 (CCK-8) (APExBIO, USA). First, cells (2 × 103 cells per well) were resuspended and plated in 96-well plates in 100 µl medium. Then, complete medium and CCK-8 were mixed at a proportion of 10:1, and 100 µl of the mixture was added to each well at planning time points (0, 24, 48, 72, and 96 h) after seeding. After incubation for 4 h, the relative absorbance was then examined at 450 nm with an enzyme immunoassay analyser (Thermo MK3, USA).
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