A 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and LDH assay were selected to evaluate the effects of DHW-221 and other drugs on cell viability. First, cells were seeded into 96-well plates at 6 × 103 cells/well. After cell adherence, cells were treated with various concentrations of DHW-221 and other drugs for the indicated times. For the MTT assay, cells were cultured with MTT (5 mg/ml) for 4 h. The supernatant was discarded, and the formed formazan crystals were dissolved by dimethylsulfoxide (DMSO). Absorbance was recorded at 490 nm using a microplate reader (Elx 800, Bio-Tek, Winooski, Vermont, USA ). The half-maximal inhibitory concentration (IC50) values were calculated by GraphPad Prism 7.0 software (San Diego, CA, USA). Importantly, the resistance index (RI) was calculated using the following formula: RI = (IC50 of A549/Taxol cells)/(IC50 of A549 cells). For the LDH assay, the cell supernatant was collected from each well and incubated with the reagent mixture according to the manufacturer’s instructions. LDH release was measured as an indicator of cytotoxicity at 450 nm by a microplate reader (Elx 800, Bio-Tek, Winooski, Vermont, USA).
Elx800
Manufactured by Agilent Technologies
Sourced in United States, Germany, United Kingdom, France, China, Japan, Canada, Austria, Hong Kong
The ELx800 is a microplate reader designed for absorbance-based assays. It can measure the optical density of samples in microplates, which is a commonly used technique in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Methyl thiazolyl tetrazolium (mtt), by Merck Group (73 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (58 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (33 mentions)
Cck 8, by Dojindo Laboratories (24 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (18 mentions)
Cell counting kit 8 (cck8), by Beyotime (18 mentions)
Mtt solution, by Merck Group (18 mentions)
96 well plate, by Corning (17 mentions)
Cck 8, by Beyotime (16 mentions)
Cck 8 assay, by Dojindo Laboratories (16 mentions)
Prism 5, by GraphPad (13 mentions)
Celltiter 96 aqueous one solution cell proliferation assay, by Promega (12 mentions)
Cck 8 solution, by Dojindo Laboratories (11 mentions)
Cck 8 kit, by Dojindo Laboratories (11 mentions)
Cell counting kit 8 cck 8 assay, by Dojindo Laboratories (10 mentions)
Mtt reagent, by Merck Group (10 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (10 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (10 mentions)
Cck 8, by Agilent Technologies (9 mentions)
Crystal violet, by Merck Group (8 mentions)
2 169 protocols using elx800
1
Evaluating Cell Viability with MTT and LDH Assays
2
Antioxidant Activities in SH-SY5Y Cells
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The SH-SY5Y cells were seeded into 6-well plates and pretreated with 8 at 10, 20, and 40 μM and rosiglitazone (40 μM) for 10 h, followed by treatment with 650 μM H2O2 for an additional 14 h. After treatment, the cells were collected and lysed in the lysis buffer for 30 min on ice. The SOD and CAT activities were measured according to the manufacturer’s instructions of EZ-Catalase assay kit (DoGenBio Co., Ltd., Seoul, Korea) and the Superoxide dismutase assay kit was used (DoGenBio Co., Ltd., Seoul, Korea). SOD inhibition activity was determined at 450 nm using a microplate reader (Elx 800, Bio-Tek, Winooski, VT, USA). CAT reacted with H2O2 to produce water and oxygen and the unconverted H2O2 reacted with OxiRedTM to generate a product measured at 570 nm using a microplate reader (Elx 800, Bio-Tek, Winooski, VT, USA).
3
Soil Enzyme Activity Measurement
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Urease activity (EC 3.5.1.5) was analyzed by 2 h incubation of a reaction mixture of 5.0 g of field-moist soils and 2.5 mL of 80 mM urea solution at 37 °C (Kandeler and Gerber 1988 ). Deionized water was added to the controls. The ammonium released was extracted with 50 mL KCl solution and product was measured at 690 nm with a digital UV–Vis spectrophotometer (Biotek ELx800) against the reagent blank. Urease activity was expressed as μg ammonium g−1 soil 2 h−1. Acid phosphatase (EC 3.1.3.2) was assayed by a colorimetric method of Tabatabai and Bremner (1969) with the exception of using a homogenized reaction of 1.0 mL 25 mM p-nitrophenol phosphate (substrate), 4.0 mL Modified Universal Buffer and 0.25 mL toluene, and that the product p-nitrophenol was extracted with 4.0 mL of 0.5 M NaOH at pH 6.5. The p-nitrophenol released was measured at 410 nm with a digital UV–Vis spectrophotometer (Biotek ELx800) and activity expressed as μg p-nitrophenol g-1 soil h−1. One control and two replicates from each soil were used in the analyses of urease and phosphatase. Enzyme activity was expressed on a moisture-free basis. Moisture content of soil was calculated from weight lost after drying at 105 °C for 24 h.
4
Colorimetric Assays for Cell Viability and Death
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Cell viability was determined using a cell counting kit (CCK‐8; Dojindo Laboratories). In brief, 10 μL of CCK‐8 was incubated with neuronal cultures for 2 h at 37°C in the dark, according to the manufacturer's instructions. A microplate reader (ELX800; BioTek) was then used to detect absorbance at 450 nm, which reflects cell viability. The cell viability was calculated as follows: cell viability (%) = (ODexperimental–ODblank)/(ODCON–ODblank) × 100%.
Cell death was determined by lactic dehydrogenase (LDH) release using the LDH assay kit (Abcam Inc.). In brief, 100 μL LDH reaction mix was added to each well and incubated for 30 min. The absorbance values were measured at 490 nm on the microplate reader (ELX800; BioTek). Results were normalized to the control group, the amount of LDH release of which was considered as 100%.
Cell death was determined by lactic dehydrogenase (LDH) release using the LDH assay kit (Abcam Inc.). In brief, 100 μL LDH reaction mix was added to each well and incubated for 30 min. The absorbance values were measured at 490 nm on the microplate reader (ELX800; BioTek). Results were normalized to the control group, the amount of LDH release of which was considered as 100%.
5
Quantification of Phenolic and Flavonoid Content
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Total phenolic content (TPC) was determined spectrophotometrically using the Folin-Ciocalteu reagent, according to the methodology described by Mazzucotelli et al. [10 (link)]. The absorbance was measured at 750 nm in a spectrophotometer (ELx800, Biotek, USA) after 2 h of incubation. Results were expressed as mg of gallic acid equivalents (GAE)/100 g FW.
Total flavonoids content (TFC) of bioaccessible fraction or ethanolic extract was quantified by following the methodology described by Mazzucotelli et al. [10 (link)]. The absorbance was read at 496 nm (ELx800, Biotek, USA). The results were expressed as mg of quercetin equivalents (QE)/100 g FW.
Total flavonoids content (TFC) of bioaccessible fraction or ethanolic extract was quantified by following the methodology described by Mazzucotelli et al. [10 (link)]. The absorbance was read at 496 nm (ELx800, Biotek, USA). The results were expressed as mg of quercetin equivalents (QE)/100 g FW.
6
Evaluating iNSC Viability on PLGA-PEG Scaffolds
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Cell viability on PLGA-PEG scaffolds was evaluated by Cell Counting Kit-8 (Nanjing, KeyGEN Biotech Co., Ltd., China) according to the manufacturer’s procedure. Briefly, after cultured for 2 days, 100μl CCK-8 solution was added to each well (n = 3) and OD of the solution was measured at 450 nm (Elx800, Biotek, USA) after incubated for 2 h at 37°C.
iNSCs (1×104 per well) were seeded onto PLGA-PEG scaffolds in 96-well plates. After incubation for 3, 6 and 9 hours at 37°C, scaffolds were washed in PBS for three times to remove the cells that did not adhere to the scaffolds. The remaining cells were collected with 0.25% trypsin with EDTA and counted under inverted optical microscope (NIKON TS100, Japan).
Cell proliferation in scaffolds was evaluated by Cell Counting Kit-8 (Nanjing, KeyGEN Biotech Co., Ltd., China) 1, 2, 3, 5, 7, and 9 days after seeding. Scaffolds were incubated with CCK-8 solution for 2 h at 37°C and OD of the solution was measured at 450 nm with the 96-well plates (Elx800, Biotek, USA). The proliferation curve of iNSCs on scaffolds was drawn.
iNSCs (1×104 per well) were seeded onto PLGA-PEG scaffolds in 96-well plates. After incubation for 3, 6 and 9 hours at 37°C, scaffolds were washed in PBS for three times to remove the cells that did not adhere to the scaffolds. The remaining cells were collected with 0.25% trypsin with EDTA and counted under inverted optical microscope (NIKON TS100, Japan).
Cell proliferation in scaffolds was evaluated by Cell Counting Kit-8 (Nanjing, KeyGEN Biotech Co., Ltd., China) 1, 2, 3, 5, 7, and 9 days after seeding. Scaffolds were incubated with CCK-8 solution for 2 h at 37°C and OD of the solution was measured at 450 nm with the 96-well plates (Elx800, Biotek, USA). The proliferation curve of iNSCs on scaffolds was drawn.
7
Rezazurin-Based Cell Viability and Collagen Deposition Assay
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Rezazurin (5 µg/ml in PBS) was used to assess cellular viability. Media were removed from constructs followed by incubation in 2.5 ml of Rezazurin for 1 h at 37°C. Rezazurin (200 µl) was taken from each sample and absorbance at 570 nm measured (Bio-tek ELx800). For collagen deposition, 2 ml of 1 mg/ml Sirius red (F3B (C.I. 35,780, Direct Red 80, Sigma–Aldrich, Dorset, UK) dissolved in saturated picric acid was added to each fixed construct and shaken for 18 h at room temperature. After prolonged washing with PBS (no further red color was eluted), the remaining stain from scaffolds was eluted by incubating constructs in 500 μl of destaining solution (0.2 M NaOH/methanol (1:1)) for 30 min. Samples (100 μl) were taken and absorbance was measured at 490 nm (Bio-tek ELx800).
8
Cytotoxicity and Hemocompatibility Assessment of CFCP
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The cytotoxicity of CFCP to DCs and neutrophils was assessed using the CCK-8 kits (Beyotime, China). Briefly, cells were inoculated into 96-well plates (1 × 104 per well) and cultured overnight. Different concentrations of CFCP (0, 20, 30, 50, 100, and 200 μg/ml) were added to the culture medium, and incubation was continued for 24 h. Subsequently, cells were washed with PBS to remove the excess NPs and incubated with a DMEM medium containing 10 % CCK-8 reagent for 30 min. Finally, the absorbance of each culture system was measured at 450 nm using a microplate reader (BioTek ELX800, USA).
The hemolysis assay was used to assess the hemocompatibility of CFCP. Fresh blood from mice was collected, centrifuged, and resuspended in a PBS solution containing heparin. After the different treatments, the blood cell suspension was incubated at 37 °C for 2 h. The supernatant was collected and the absorbance at 540 nm was measured using a microplate reader (BioTek ELX800, USA).
The hemolysis assay was used to assess the hemocompatibility of CFCP. Fresh blood from mice was collected, centrifuged, and resuspended in a PBS solution containing heparin. After the different treatments, the blood cell suspension was incubated at 37 °C for 2 h. The supernatant was collected and the absorbance at 540 nm was measured using a microplate reader (BioTek ELX800, USA).
9
Colorimetric Assays for Lipid Peroxidation
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MDA concentration was assayed colorimetrically using the thiobarbituric acid reactive substances (TBARS) method with 1,3,3,3-tetraethoxypropane (Sigma-Aldrich, Saint Louis, MO, USA) as a standard [25 (link)]. The absorbance was measured at 535 nm with microplate reader ELx800 and Gen5 2.01 software (BioTek Instruments, Winooski, VT, USA).
4-HNE concentrations was measured using a commercial enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (Cell Biolabs, Inc., San Diego, CA, USA, and USCN Life Science). The absorbance was measured at 405 nm with microplate reader ELx800 and Gen5 2.01 software (BioTek Instruments, Winooski, VT, USA).
4-HNE concentrations was measured using a commercial enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (Cell Biolabs, Inc., San Diego, CA, USA, and USCN Life Science). The absorbance was measured at 405 nm with microplate reader ELx800 and Gen5 2.01 software (BioTek Instruments, Winooski, VT, USA).
10
RNA Extraction from BDC Cells
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BDC cells were lysed by adding 1 ml of Tri-reagent (Sigma-Aldrich; Merck KGaA) over 1 min. The lysate was the treated with 200 µl chloroform (Sigma-Aldrich; Merck KGaA) and incubated for 10 min. It was then centrifuged at 12,000 × g at 4°C for 10 min. An equal volume of isopropanol (Sigma-Aldrich; Merck KGaA) was treated to the supernatant and incubated for 15 min. The final product was centrifuged again at 12,000 × g at 4°C for 15 min. The pelleted RNA was rinsed with 70% ethanol and 50 µl nuclease-free-water (Roche Diagnostics) was added to dissolve the RNA, which was quantified using ELX800 (Biotek) at 260 nm.
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