The supernatant containing the lysates of rat brain tissues and NSCs was homogenized in ice-cold mitochondria isolation buffer. The total ROS level in the brain tissue was detected using the Total ROS Activity Assay kit (Sigma-Aldrich; Merck KGaA). The supernatant lysate was incubated with Total ROS Green in an incubator (5% CO2) for 6 h at 37°C. Fluorescence intensity was then detected at 525 nm emission and 490 nm excitation. The levels of malondialdehyde (MDA), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) in the rat brain tissues and NSCs were measured using standard assay kits, including the Total SOD Activity Detection kit (Solarbio, Beijing, China), Malondialdehyde (MDA) Content Detection kit (Solarbio), and Glutathione Peroxidase (GSH-Px) Detection kit (R&D Systems, Inc., Minneapolis, MN, USA), according to the manufacturer's instructions. The OD value was then measured using a LUX microplate reader (Thermo Fisher Scientific).
Lux microplate reader
Manufactured by Thermo Fisher Scientific
Sourced in United States
The LUX microplate reader is a versatile instrument designed for sensitive and accurate fluorescence-based measurements in a microplate format. It offers a core function of detecting and quantifying fluorescent signals from samples in a 96-well or 384-well microplate.
Automatically generated - may contain errors
Lab products found in correlation
96 well black walled clear bottomed plate, by Corning (2 mentions)
Skanit 5, by Thermo Fisher Scientific (2 mentions)
Kaleidagraph, by Synergy Software (2 mentions)
Malondialdehyde mda content detection kit, by Solarbio (1 mentions)
Kga311, by Keygen Biotech (1 mentions)
Kgt5131, by Keygen Biotech (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
Antimycin a, by Merck Group (1 mentions)
Rotenone, by Merck Group (1 mentions)
Luria broth lb, by Thermo Fisher Scientific (1 mentions)
Lb agar, by Thermo Fisher Scientific (1 mentions)
Bright glo, by Promega (1 mentions)
2100 bioanalyzer instrument, by Agilent Technologies (1 mentions)
Nanodrop lite spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Drop plate, by Thermo Fisher Scientific (1 mentions)
Annexin 5 fitc apoptosis kit, by Beyotime (1 mentions)
Facsaria 2 flow cytometer, by BD (1 mentions)
Cck 8 assay, by Merck Group (1 mentions)
L 012, by Fujifilm (1 mentions)
Horseradish peroxidase, by Merck Group (1 mentions)
23 protocols using lux microplate reader
1
Oxidative Stress Evaluation in Rat Brain
2
Cell Viability Assay of ART and ART@LIP
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To detect the cell viability,
the B-CPAP cells were seeded into a 96-well plate and cultured overnight.
The cells were treated with 200 μM ART or ART@LIP and co-treated
with 50 μM DFO for 24 h. After treatment, the culture medium
was removed, and the Alamar Blue work solution (40 μM resazurin
sodium in medium) was added into wells. After incubation for 2 h,
the fluorescence intensity of resorufin of each well was detected
on a Varioskan LUX microplate reader (ThermoFisher Scientific, USA).
B-CPAP cells (4 × 104) were seeded into a 96-well
flat clear bottom black microplate (Corning, USA) and cultured overnight.
Upon treatment with 200 μM ART or ART@LIP for 24 h, the culture
medium was removed, and the cells were stained by the Calcein/PI working
solution (Calcein AM and PI were diluted with PBS at a ratio of 1:1000;
Beyotime, China) followed by incubation at 37 °C in the dark
for 30 min. After incubation, the fluorescence intensity of Calcein
(Ex/Em = 494:517 nm) and PI (535/617 nm) was measured on a Varioskan
LUX microplate reader. The ratios of the fluorescence intensity of
Calcein/PI of each well were then analyzed.
the B-CPAP cells were seeded into a 96-well plate and cultured overnight.
The cells were treated with 200 μM ART or ART@LIP and co-treated
with 50 μM DFO for 24 h. After treatment, the culture medium
was removed, and the Alamar Blue work solution (40 μM resazurin
sodium in medium) was added into wells. After incubation for 2 h,
the fluorescence intensity of resorufin of each well was detected
on a Varioskan LUX microplate reader (ThermoFisher Scientific, USA).
B-CPAP cells (4 × 104) were seeded into a 96-well
flat clear bottom black microplate (Corning, USA) and cultured overnight.
Upon treatment with 200 μM ART or ART@LIP for 24 h, the culture
medium was removed, and the cells were stained by the Calcein/PI working
solution (Calcein AM and PI were diluted with PBS at a ratio of 1:1000;
Beyotime, China) followed by incubation at 37 °C in the dark
for 30 min. After incubation, the fluorescence intensity of Calcein
(Ex/Em = 494:517 nm) and PI (535/617 nm) was measured on a Varioskan
LUX microplate reader. The ratios of the fluorescence intensity of
Calcein/PI of each well were then analyzed.
3
Cell Viability Assay by MTT
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The viability of cells after being cultured for 24 h, 48 h, and 72 h was determined by MTT assay. Concretely, 6 × 103 cells were seeded into each well of the 96-well plates and cultured for 24 h, 48 h, and 72 h. Then, 50 μl 1× MTT buffer (KGA311, KeyGEN BioTECH, Nanjing, China) was added to each well to further incubate the cells for 4 h. After the medium in each well was removed, 150 μl of dimethyl sulfoxide (DMSO; KGT5131, KeyGEN BioTECH) was put into each well, subsequent to which the optical density (OD) value at a wavelength of 490 nm was detected by a Varioskan LUX Microplate reader (Thermo Scientific).
4
Plasma BNP Quantification Using EIA
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Plasma BNP concentration was measured by using a Brain Natriuretic Peptide EIA Kit (RAB0386-1KT, Sigma-Aldorich, Inc., St. Luise, MO, USA) according to the procedural manual. In brief, the samples and standards mixed with 10 pg/ml of biotinylated BNP peptide were added to the anti-BNP antibody-immobilized plate, where the biotinylated BNP peptide competed with unlabeled (endogenous) BNP for binding to the antibody. After incubating plate at 4°C overnight and washing wells, horseradish peroxidase (HRP)-streptavidin solution was added to each well, and incubated for 45 minutes at room temperature. After washing, tetramethylbenzidine (TMB) solution was then added to each well to visualize the immunoreaction. After adding stop solution, absorbance of each well was measured by using Varioskan LUX microplate reader (ThermoFisher) at 450nm. The result was obtained from one measurement (n = 4 in each group) performed in duplicate.
5
Organoid Cytokine and Collagen Analysis
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Organoid culture samples were obtained after infection, centrifuged at 1,000 g for 20 min, and stored at −80°C for future experiments. ELISA assay kits were applied to determine the levels of TGF-β1, IL-13, IL-33, and collagen type I alpha-1, according to the manufacturer's protocols (Cloud-Clone Corp., China). All samples were detected once using their corresponding kit with duplicate samples. Optical density (OD) was read at 450 nm for all indices, using a Varioskan LUX microplate reader (Thermo Scientific) with SkanIt Software 6.0.2.
6
Redox Stress Monitoring in Cells
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2 × 104 cells/well were plated into a black‐walled, clear‐bottom 96 well plate (Nunc). Cells were incubated with either 5 μm of 2,7‐dichlorodihydruofluroescein diacetate (H2DCFDA) or 5 μm MitoSOX™ Red (Thermo Fisher Scientific, Waltham, MA, USA, M36008) in Hanks Balanced Salt Solution (HBSS) for 30 min at 37 °C. Cells were then incubated with or without 2 μm Rotenone (Sigma) or 4 μm antimycin A (Sigma) and scanned using a Varioskan LUX microplate reader (Thermo Fisher Scientific VL0LATD0) at 37 °C. Fluorescence was measured every 5 min using excitation/emission wavelengths of 485/520 nm for H2DCFDA and 510/600 nm for MitoSOX™. Significant differences were determined using Student's two‐tailed t‐tests.
7
Sulforhodamine B Assay for Cell Proliferation
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Cell proliferation was determined by the sulforhodamine B assay (SRB). Cells were seeded on 96-well plates at a density of 1 × 104 cells/ml per well. Before the addition of the oil emulsions, one plate from each cell line was fixed with cold 50% trichloroacetic acid (TCA) at 4-8°C (these plates were the control). After 48-hour incubation with increasing concentrations of oil emulsion, the culture plates were also fixed with 50% TCA for 1 h at 4-8°C. The plates were then washed five times under running water, the plates were air-dried, and then, sulforhodaminen B dye was added for another 30 minutes to stain cell proteins. Unbound dye was removed five times by washing with 1% acetic acid and air-dried again. The SRB dye was dissolved in Trizma solution, and the absorbance was measured at 570 nm, using a Varioskan LUX microplate reader (Thermo Scientific, USA).
8
Growth of S. aureus Strains
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S. aureus strains, SH1000 and SH1000 Δfnb, were kindly provided by Simon Foster (University of Sheffield, Sheffield, United Kingdom). MRSA isolates were retrieved from skin infections of patients at Northern General Hospital, Sheffield, and kindly provided by Sue Whittaker. Liquid cultures were grown in Luria broth (LB; Oxoid, Ltd., Basingstoke, United Kingdom) microaerobically at 37°C in a humidified atmosphere with constant agitation. OD600nm readings were taken using a Varioskan LUX microplate reader (ThermoFisher Scientific, Waltham, MA, USA). Solid cultures were grown on LB agar (Oxoid) overnight at 37°C. Freshly grown plates were used to inoculate all liquid cultures.
9
Measuring Intracellular Nitric Oxide
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For the evaluation of intracellular concentration of nitric oxide, Griess reagent (1 : 1 mixture (v/v) of 1% sulfanilamide in 5% phosphoric acid and 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride) was added to the supernatant plates. The plates were left for 20 minutes in the dark at RT. Nitrite level was measured at 548 nm using a Varioskan LUX microplate reader (Thermo Scientific, USA) [29 (link)].
10
Quantifying Intracellular ROS Levels
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Detection of intracellular ROS level was performed using the fluorescent dye 2′,7′-dichlorofluorescein diacetate (H2DCF-DA). After the metal treatment, cells were washed twice with 50 mM potassium phosphate buffer, and the dye was added in a final concentration of 10 µM. An incubation time of 30 min in the dark and another washing step were employed before the dye fluorescence (ex/em = 488/520 nm) was measured with a Varioskan LUX microplate reader (Thermofisher, Waltham, MA, USA). To determine cell viability, cell suspensions were properly diluted with PBS to a dilution of 10−6 and plated on solid YPD plates. After 48 h incubation at 28 °C, yeast colonies formed on the solid YPD plates and were counted. The number of formed yeast colonies was expressed as colony forming units (CFU). The results were expressed as the fluorescence divided with values for the cell viability to obtain more accurate results.
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