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Microplate reader

Manufactured by Anthos Labtec
Sourced in Austria

A microplate reader is a laboratory instrument designed to measure and analyze the optical properties of samples contained in a microplate. It is used to quantify the absorbance, fluorescence, or luminescence of liquid samples within the wells of a microplate. The microplate reader provides accurate and reproducible data for a variety of applications, such as enzyme-linked immunosorbent assays (ELISA), cell-based assays, and high-throughput screening.

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11 protocols using Microplate reader

NIH3T3 cells in 96-well culture plates were transfected with siRNAs or cDNA constructs and incubated for 24 – 72 hr. At each designated time point after transfection, culture media were removed, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) (0.5 mg/ml) was added, and cells were incubated at 37°C for 2 hr in a CO2 incubator. After dissolving the insoluble crystals that formed in DMSO, absorbance was measured at 570 nm using a microplate reader (Anthos Labtec Instruments, Wals, Austria).
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Cell viability was determined by 3-(4,5-dimet hylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Hypothalamic neuronal GT1-7 cells were seeded in triplicate at a density of 1 × 104 cells per well on a 96-well plate. After treatment, culture media were removed and MTT (0.5 mg/mL) added, followed by incubation at 37 °C for 2 h in a CO2 incubator. After dissolving the insoluble crystals that formed in DMSO, absorbance was measured at 570 nm using a microplate reader (Anthos Labtec Instruments, Wals, Austria).
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Astrocyte cultures were treated with stimuli in 96‐well plates, and then NO2 in culture media was measured in order to assess NO production levels by the Griess reaction as described previously.24 Sample aliquots of 50 μl were mixed with 50 μl of Griess reagent (1% sulfanilamide, 0.1% naphthylmethyl diamine dihydrochloride, 2% phosphoric acid) in 96‐well plates, and then incubated at 25℃ for 10 min. The absorbance at 540 nm was measured using a microplate reader (Anthos Labtec Instruments). NaNO2 was used as a standard to calculate NO2 concentration.
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Cell viability was measured by using CCK-8 assay (Dojindo, JAPAN). After transfected with Ano1 or empty vectors, MCF7 or MDA-MB-435S cells (8,000 cells/well) were seeded into 96-well plates (Coring, Lowell, MA). Cells were allowed to grow in the growth medium for 48 h. Cells were then incubated with WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium) for 2.5 h. Plates were read at 450 nm wavelength in a microplate reader (Anthos Labtec Instruments GmbH, Austria).
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Approximately 5 mL of fasting (for about 6 h) blood was collected, and the serum was separated for subsequent immunofluorescence measurements of PGI, PGII, and G-17. A serum pepsinogen and G-17 kit was purchased from BIOHIT Healthcare (Hefei) and a microplate reader from Anthos Labtec Instruments (Austria).
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6

Evaluating Gallic Acid's Cytotoxicity on Milk Phagocytes

A total of 1 × 105 isolated milk phagocytes were seeded in duplicate into a 96-well flat-bottom plate and subsequently incubated with either HBSS as control or various dilutions of gallic acid (12, 25, 50, 100, and 200 μM) in RPMI-1640 medium. The plate was incubated at 37°C with 5% CO2 for 1 h. Following incubation, the plate was centrifuged at 1,200 rpm for 3 min, and the supernatant was discarded. Subsequently, all wells were treated with 2 μg/mL of MTT in RPMI-1640 medium and incubated for 90 min. After centrifugation, the medium containing non-metabolized MTT was aspirated, and 150 μL of dimethyl sulfoxide (DMSO) was added to solubilize the formazan crystals formed by viable cells (3 (link)). To indicate the baseline color in the assay, the MTT solution (Blank) was added. The absorbance (OD) was then measured at a wavelength of 570 nm using a microplate reader (Anthos Labtec Instruments, Wals, Austria). The percentage of cell viability was quantified using the following equation:
The percentage of milk phagocyte cell death following treatment with various concentrations of gallic acid was calculated by subtracting the percentage viability from 100. A dose–response curve was generated, with gallic acid concentration represented on the x-axis and the percentage of milk phagocyte cell death on the y-axis.
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We followed the methods described by Kim et al. (2016) to measure cell viability [44 (link)]. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Hypothalamic neuronal GT1-7 cells were seeded at a density of 1 × 104 cells per well in a 96-well plate in triplicate. Following treatment, culture media were got rid of and MTT (0.5 mg/mL) was added, after which samples were incubated at 37°C for 2 h in a CO2 incubator. Absorbance was measured at 570 nm using a microplate reader (Anthos Labtec Instruments, Wals, Austria) after dissolving the insoluble crystals in dimethyl sulfoxide (DMSO).
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For the 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay, cells were treated with various reagents for a designated period. After treatment, MTT (0.5 mg/mL; Sigma-Aldrich) was added to the cells, which were then incubated for 2 h at 37 °C in a 5% CO2 incubator. Subsequently, dimethyl sulfoxide (DMSO) was added to dissolve the insoluble crystals and the absorbance was measured at 570 nm using a microplate reader (Anthos Labtec Instruments, Wals, Austria).
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The proliferation ability of SGC-7901 cells was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich, St. Louis, MO, USA) assay. Approximately 104 cells were seeded into each well of 96-well plates. Then, SGC-7901 cells were transfected with pcDNA3.1+HA-FOXM1, pcDNA3.1+HA empty vector, pcDNA3.1+FOXM1-siRNA or pcDNA3.1+NC-siNRA following the manufacturer's instructions. After 6 h of transfection, the cells were treated with TSN or a placebo. After 12, 24 or 48 h of incubation, 25 µl of MTT (5 mg/ml) was added to each well and the plates were incubated for 4 h at 37°C. Then, the precipitates in each well were solubilized with 150 µl of dimethyl sulfoxide (DMSO; Sigma-Aldrich), and the plates were read on a microplate reader (Anthos Labtec Instruments, Salzburg, Austria) at 490 nm. Values were normalized using the control value.
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Protein extracts were obtained from samples by tissue homogenization in lysis buffer (Cloud-Clone Corp., USA) and centrifugation for 10 min at 2590×/g at 4 °C followed by 5 min at 5000×/g at 4 °C according to the manufacturer’s instructions. Protein concentration was determined by Bradford protein assay kit (Bio-Rad, Germany). Enzyme-linked immunosorbent assay (ELISA) was performed to quantify the protein levels of GRN, NOTCH3, FN1, and PINK1. Standards, samples, reagents, and microplate preparations were performed as described by the manufacturer. Optical density of each well was measured using a microplate reader at a wavelength of 450 nm (Anthos Labtec Instruments, Austria). All conditions were measured in duplicates. Protein concentrations were calculated using the mean of the duplicates in relation to the total protein amount. The following kits were used: Progranulin ELISA Kit (No. E-EL-H1578, Elabscience, USA), Human Notch Homolog 3 ELISA Kit (No. SEL147Hu, Cloud-Clone Corp., USA), Quantikine ELISA Human Fibronectin (No. DFBN10, R&D Systems, USA), Human PINK1 ELISA Kit (No. MBS9327222, MyBioSource, USA).
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