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Microplate reader

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The Microplate reader is a laboratory instrument used to measure the absorbance, fluorescence, or luminescence of samples in a microplate format. It is designed to quantify and analyze a wide range of biological and chemical assays, such as enzyme-linked immunosorbent assays (ELISA), cell-based assays, and other microplate-based applications.

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6 925 protocols using Microplate reader

Oxidative stress-related enzymes, including superoxide dismutase (SOD), malondialdehyde (MDA) and catalase (CAT), were analyzed in vivo and in vitro. The activity of SOD was measured using a SOD assay kit (#706003; Cayman Chemical Company). The activity of SOD was analyzed using a microplate reader (BioTek Instruments, Inc.) at 450 nm. The activity of MDA was measured using a MDA assay kit (#700870; Cayman Chemical Company). The activity of MDA was analyzed using a microplate reader (BioTek Instruments, Inc.) at 405±414 nm. The activity of CAT was measured using a CAT assay kit (#707002, Cayman Chemical Company). The activity of CAT was analyzed using a microplate reader (BioTek Instruments, Inc.) at 340 nm.
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Glucose uptake was measured using a glucose uptake colorimetric assay kit (BioVision) according to the manufacturer’s protocol. Briefly, cells were seeded at a density of 2,000 cells per well in a 96-well plate. Cells were starved in serum free culture medium overnight. Cells were then washed with phosphate-buffered saline (PBS) and incubated with 100 μL Krebs-Ringer-Phosphate-HEPES (KRPH) buffer containing 2% BSA for 40 min. Ten microliters of 10 mM 2-deoxyglucose was then added and incubated for 20 min. Cells were proceeded to oxidation step to generate NADPH, which is determined by an enzymatic recycling amplification reaction. The absorbance at 412 nm was recorded using a BioTek microplate reader and normalized to protein concentration.
The culture medium was collected for the measurement of lactate release from cells. Lactate levels were determined using a lactate colorimetric assay kit (BioVision) according to the manufacturer’s protocol. The absorbance at 450 nm was recorded using a BioTek microplate reader and normalized to protein concentration.
ATP and ADP levels were measured using an ATP/ADP colorimetric assay kit (BioVision) according to the manufacturer’s protocol. The absorbance at 450 nm was recorded using a BioTek microplate reader and normalized to protein concentration.
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For the melanin quantification assay, the cells were pre-incubated with 200 nM α-melanocyte-stimulating hormone (α-MSH; Sigma-Aldrich; Merck KGaA) for 1 h at 37°C before adding the essential oils to promote melanin production. The assigned tested essential oils at non-toxic concentrations (Table II) or anti-melanogenic agent arbutin (250 µM) were added to the culture medium and incubated for a further 72 h at 37°C. α-MSH was used without essential oils as a positive control. DMSO alone was used as a standard control. Following treatment, the extracellular melanin content in 200 µl of culture media was measured using a microplate reader using a microplate reader (Agilent Technologies, Inc.) at 405 nm. To measure the intracellular melanin content, the cells were washed twice with phosphate-buffered saline (PBS) and collected using trypsinization. Centrifugation at 20,000 × g for 15 min at 4°C was performed, and the melanin pellets were dissolved in 1 N NaOH containing 10% DMSO for 1 h at 60°C. The mixed homogenate (100 µl) was placed in a 96-well microplate, and the OD values were measured using a microplate reader (Agilent Technologies, Inc.) at 405 nm. The extracellular and intracellular melanin contents per well were calculated and expressed as a percentage of the control.
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Assay kits for total protein (TP), albumin (ALB), glucose (GLU), and uric acid (UA), provided by Nanjing Institute of Biological Engineering (Nanjing, China), were used to measure the concentrations of components in the plasma samples. The component concentrations in the separated plasma were determined by the microplate reader (Bio Tek, Winooski, VT, USA). Plasma sample processing was performed according to the instruction by the supplier. For the measurement, 150 µL of the mixture of the plasma sample and the reaction reagent were pipetted into 96-well plate. The absorbance measurements were carried out at the corresponding wavelength with the microplate reader (Bio Tek, Winooski, VT, USA). For comparison, the concentrations of components in plasma samples prepared by the common centrifuge technique were assessed.
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CHO-IR cells were seeded in a 96-well plate with 15,000 cells per well in 100 μL of complete medium. Cells were incubated overnight, followed by 15 min of treatment with different concentrations of drugs. The cell lysate (100 μg) then was incubated with 4 mmol/L p-nitrophenyl phosphate, 50 mmol/L Tris-HCl (pH 7.5), 100 mmol/L NaCl, and BSA (0.1 mg/mL) for 20 min at 37°C. The reaction was terminated by the addition of 1 mol/L NaOH. The p-nitrophenol formed was determined spectrophotometrically at 400 nm using a BioTek microplate reader (Highland Park, Vermont).
For in vitro PTP1B assay, different concentrations of drugs were incubated with PTP1B (0.4 μg) and 4 mmol/L p-nitrophenyl phosphate in 50 mmol/L potassium phosphate buffer (pH 7.5), containing 0.1 mmol/L EDTA. Incubations were carried out for 30 min at 37°C and terminated by the addition of NaOH. The p-nitrophenol formed was determined at 400 nm using a BioTek microplate reader.
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The CCK-8 assay was used to detect the proliferation of the HCT116/ATB and LOVO/ATB cells. Briefly, 100 μL of cell suspension was inoculated in a 96-well plate (1 × 103 cells/well) and was cultured at 37°C, 5% CO2. After that, 10 μL of CCK-8 solution (Sigma-Aldrich) was added to the cell culture at 0, 24, 48, and 72 hours. Finally, the optical density value was recorded at 450 nm using a Microplate reader (BioTek, Winooski, VT, USA).
To analyze the cytotoxicity of ATB to HCT116/ATB and LOVO/ATB cells, different concentrations of ATB were added to the cell culture. Then, the plate was cultured for 48 hours at 37°C, 5% CO2. Then, 10 μL of CCK8 solution was added to each well, and the cells were further cultured for 1–4 hours. The cytotoxicity of ATB to HCT116/ATB and LOVO/ATB cells was measured using a Microplate reader (BioTek).
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For the cell viability assay, 48 h after transduction of A-172 and U251 cells approximately 5000 (cells/well) of controls and transduced cells were seeded in 96-well plates. Then at 72, 96 and 120 h post-transduction the cells were treated with 10 µl MTT reagents (5 mg/ml) (MTT, Sigma, St. Louis, USA) per well and maintained for 3 hours at 37 °C. Then the culture media were discarded, and 100 µl of DMSO was added to each well; furthermore absorbance was measured at 570 nm with a microplate reader (BioTek, Winooski, USA). All tests were performed in triplicate. The proliferation assay was done 48 h after transduction of A-172 and U251 cells. Approximately 1.5 × 104 (cells/well) were cultured in 24-well plates and cultured at 37 °C in a 5% CO2 incubator. Then at 72, 96, 120 and 168 h post-transduction, the cells were fixed for 30 min in methanol and stained with 0.1% crystal violet. Stained cells were rinsed twice with PBS and allowed to air-dry. Ethanol was then added to dissolve the crystal violet. Absorbance was measured at 570 nm with a microplate reader (BioTek, Winooski, USA). All tests were performed in triplicate.
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Nitrite was measured in serum and heart hemogenate by Griess reagent method using a standard enzyme-linked immunosorbent assay (ELISA) kit (Promega Corp., USA, Cat#G2930). In brief, 100 μl of serums or tissue homogenates were added to a 96-well flat-bottomed microplate. Then, sulfanilamide solution and N-1-naphtylethylenediamine dihydrochloride under acidic conditions were added to all collected samples, respectively. The absorbance was detected by a microplate reader (Biotek, USA) in 520–550 nm wavelengths. The limit detection was 2.5 μM nitrite.[16 (link)17 (link)]
Serum and heart tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) were quantified by available ELISA kits (eBioscience Co., San Diego, CA, USA) according to manufacturers’ instruction. The absorbance was measured using a microplate reader (Biotek, USA) and concentration of each factor was calculated by comparison curve established in the same measurement. Each cytokine assay was performed in duplicate each time.
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The concentrations of TNF-α, IL-1β, IL-6, MCP-1, MIP-2, IFN-γ and MPO in the liver tissue samples were measured by a Mouse ELISA Kit (Jiangsu Yutong Biological Technology Co) with a microplate reader (Bio Tek, USA). The concentration of NO as well as the enzyme activity of ALT and AST in the liver tissue samples were respectively measured by a Nitric Oxide (NO) assay kit (Microwell plate method), Alanine aminotransferase Assay Kit and Aspartate aminotransferase Assay Kit (Nanjing Jiancheng Bioengineering Institute) with a microplate reader (Bio Tek, USA).
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The LDH release assay was used to determine the membrane integrity for intracellular LDH would release to the extracellular media if the cellular membrane was damaged31 (link). The Cytotoxicity Detection Kit was purchased from Roche Applied Science and the reaction mixture was prepared according to the manufacturer’s instructions. After exposed to CuO NPs for 24 h, the culture supernatants were obtained by centrifugation (12000 rpm for 3 min) and seeded in a 96-well plate, and then 50 μL of reaction mixture was added for 30 min incubation at 30 °C. Finally, the data were obtained at 490 nm absorbance via a microplate reader (BioTek, USA). Cell viability was detected by Cell Counting Kit-8 (CCK-8, Dojindo) according to the manufacturer’s instructions. In detail, 100 μL of the cell suspensions were obtained after 24 h exposure and placed in a 96-well plate, following by seeding 10 μL of CCK-8 for 30 min at 30 °C. Thereafter, the absorbance was recorded at 450 nm via a microplate reader (BioTek, USA).
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