Cell viability assays were performed in 96-well plates using Thiazolyl Blue Tetrazolium Blue reagent (MTT) (Sigma Aldrich, MO, USA) and measured at 450 nm in a Dynex MRX Revelation Microplate Reader (Chantilly, VA). Readings were plotted in Excel Microsoft software; experimental groups were standardized to non-infected and non-treated controls using the following equation: Results were reported as the percent viability (%) from control. Bicinchoninic acid assays for total protein quantitation from MDM supernatants were performed according to manufacturer’s instructions (Bio Rad, La Jolla, CA). Samples from different donors were assayed in triplicates and read at 450nm in a Dynex MRX Revelation Microplate Reader (Chantilly, VA).
Mrx revelation microplate reader
Manufactured by Dynex
Sourced in United States
The MRX Revelation microplate reader is a versatile laboratory instrument designed for absorbance-based measurements. It can accurately quantify and analyze the optical density of samples in microplates. The device features a wide measurement range and supports multiple wavelength selections to accommodate a variety of assay types.
Automatically generated - may contain errors
Lab products found in correlation
Thiazolyl blue tetrazolium blue reagent mtt, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Sodium bicarbonate, by Merck Group (1 mentions)
Serum separator tube, by Greiner (1 mentions)
8 protocols using mrx revelation microplate reader
1
Quantifying Cell Viability and Protein
2
Cell Viability and Protein Quantification
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Cell viability assays were performed in 96-well plates using Thiazolyl Blue Tetrazolium Blue reagent (MTT) (Sigma Aldrich, MO, USA) and measured at 450 nm in a Dynex MRX Revelation Microplate Reader (Chantilly, VA). Readings were plotted in Excel Microsoft software; experimental groups were standardized to non-infected and non-treated controls using the following equation: . Results were reported as the percent viability (%) from control. Bicinchoninic acid assays for total protein quantitation from MDM supernatants were performed according to manufacturer’s instructions (Bio Rad, La Jolla, CA). Samples from different donors were assayed in triplicates and read at 450 nm in a Dynex MRX Revelation Microplate Reader (Chantilly, VA).
3
Cell Viability Assay with NF-κB Inhibitors
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Cells were seeded in 96 well plates at a concentration of 40,000 cells per well for FSDC, 10,000 cells/well for MEFs, 30,000 cells/well for D10, 30,000 cells/well for BMDM and BMDC and 30,000 cells/well for 293NF-κB cells. Cells were treated with listed doses of the NF-κB inhibitors and allowed to grow for 24 hours or as described. 10 μl of MTT working solution (5 mg/ml) was added to each well and incubated for 2 hours. Excess media was removed and crystals were dissolved in 20 μl of DMSO and then diluted in dH20. Absorbance was measured at 530 nm on MRX Revelation microplate reader (Dynex Technologies). Values were then normalized to untreated controls and blank wells.
4
Cytotoxicity Assessment of ND and ND@Au
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The cytotoxicity was estimated by MTT assay using ND or ND@Au and A549 cell line. A549 cells were seeded in quantity of 4 × 105 cells/well in a 96-well microtiter plate in RPMI1640 medium (Gibco, Invitrogen, UK) 2 mM l -glutamine (Invitrogen, USA), 1.5 g/l sodium bicarbonate (Sigma, UK), 10% fetal bovine serum (Gibco/Life Technologies, Carlsbad, CA, USA) and incubated at 37 °C and 5% CO2 for 24 h. After incubation, the medium was removed. Then the cells were treated with a fresh medium containing 50 µg/ml of ND or different ND@Au concentrations (1–50 μg/ml) for 24 h. After this procedure, the medium was removed and the cells were treated for 4 h with MTT reagent (with the concentration of 2.5 mg/ml). The surviving cells converted MTT-agent to formazan, a blue-purple color when dissolved in dimethyl sulfoxide. The solution was removed and 200 μl of dimethyl sulfoxide (DMSO) was added to dissolve the formazan. The optical absorption of the treated cells and control (non-treated) cells was measured at 570 nm (O.D. 570) with ELISA reader (MRX revelation Microplate Reader, DYNEX, USA). Relative percentages of surviving cells were calculated by the dividing the absorbance of the treated cells with that of the control measured in each experiment.
5
Extraction and Quantification of Lipids and Proteins from Thrombus Samples
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Frozen thrombus samples in PBS-EDTA were thawed on ice and homogenized with the Polytron PT 1,600 E homogenizer until a delicate particulate matter was suspended in PBS-EDTA solution. To prevent heating of the solution, cycles of 20 sec of homogenization and 60 sec on the ice were performed until complete homogenization. The sample was aliquoted and frozen at −80°C until the time of lipid extraction. Homogenized samples were thawed on ice and lipid extraction was performed using the 2:1 (vol/vol) chloroform: methanol. The protocol was adjusted for STEMI thrombus samples to allow 1 ml of sample to be extracted in comparison to 100 μl of a sample as initially described by Folch et al. (15 (link)). An internal standard mixture of 9:0 PC (10 ng/μl) was added to each sample before lipid extraction. Similar to plasma samples, a portion of lipid extract was reconstituted in solvent A and analyzed by LC-MS/MS.
The total protein concentration of the homogenate sample was quantified with the Pierce Microplate BCA Protein Assay Kit by Thermo Scientific. Optical Density was read at 570 nm with the Dynex MRX Revelation Microplate Reader. Protein was quantified in micrograms per milliliter of homogenate.
The total protein concentration of the homogenate sample was quantified with the Pierce Microplate BCA Protein Assay Kit by Thermo Scientific. Optical Density was read at 570 nm with the Dynex MRX Revelation Microplate Reader. Protein was quantified in micrograms per milliliter of homogenate.
6
Measuring Cell Viability with MTT Assay
7
Quantifying HIV-1 gp120-specific IgG in Mice
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HIV-1 gp120-specific IgG in mouse serum was measured by ELISA. Immulon 4 HBX high-binding plates were coated with 100 µl of purified HIV-1 HXBc2 gp120 at a final concentration of 1 µg/ml in PBS overnight at 4°C. The plates were washed once with wash buffer (0.05% Tween-20 in PBS) and then blocked with blocking buffer (2% BSA in PBS) for 1 h at room temperature; after which, the plates were washed three times with wash buffer. Dilutions of serum samples and standard for the gp120-specific IgG measurement (mouse mAb against gp120 [3B3]) were made in blocking buffer and incubated on the plate (100 µl/well) for 1.5 h at room temperature. Samples and standard were removed, and the plate was washed four times with wash buffer. Detection antibody was diluted 1:10,000 in blocking buffer and incubated (100 µl/well) for 1 h. After four washes, TMB substrate mixture (KPL) was added at 100 µl/well for 20 min; 2 N sulfuric acid (50 µl/well) was used to stop the reaction, and the optical density was read at 450 nm on a Dynex MRX Revelation microplate reader. The linear region of the standard curve was used for quantitation.
8
Quantification of Serum Granulysin Levels
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Serum GNLY concentrations were measured using the LEGEND MAX™ Human Granulysin ELISA Kit (cat. no. 438007; BioLegend, Inc.). The sera of OA patients and controls were prepared from peripheral venous blood samples (3 ml/sample), which were taken by venipuncture in plastic Serum Separator Tubes, 3.5 ml (Greiner Bio-One GmbH), allowed to clot for 20 min at 22-25˚C, and centrifuged (1,000 x g, 10 min, at 4˚C). The supernatant (serum) was collected in Cryo.S, 2 ml, round bottom, screw-cap vial (Greiner Bio-One GmbH) and stored at -80˚C until required for ELISA. Assays were performed following the manufacturer's instructions, and the absorbance was measured using an MRX Revelation microplate reader (Dynex Technologies Inc.).
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