Enzyme-linked immunosorbent assay (ELISA) for MMP-9 and MMP-2 in BM supernatant and plasma was performed using Quantikine ELISA kits (R&D Systems, Minneapolis, MN). Plates were read using Epoch7 96-well plate reader and the kinetic function on Gen5 software. Because of high cost and low availability of commercially prepared ELISA kits for neutrophil elastase in our species, an enzymatic assay of elastase (EC 3.4.1.36) (Sigma–Aldrich, St. Louis, MO) cleavage function was adapted for use in a 96-well plate with the following modifications: 10 µL of sample (BM supernatant or plasma), 120 µL of Trizma Buffer (12.1 mg/mL, pH 8.0 at 25°C) (Sigma–Aldrich), and 20 µL of N-succinyl-(Ala)3-p-nitroanilide substrate (2 mg/mL; Sigma–Aldrich) were used per well. The pathlength for 150 µL in the 96-well Flat Bottom plate (Becton Dickinson Labware, Franklin Lakes, NJ) was experimentally determined and used to correct the millimolar coefficient (for 1 cm pathlength) provided in the protocol defined as units per milliliter. All samples were run in duplicate.
Flat bottom plate
Manufactured by BD
The Flat Bottom Plate is a laboratory equipment designed for a variety of applications. It features a flat bottom surface that provides a stable platform for various experiments and procedures. The core function of this product is to hold and support samples, reagents, or other materials during laboratory work.
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Lab products found in correlation
2 protocols using flat bottom plate
1
Quantifying MMP-9, MMP-2, and Neutrophil Elastase
2
MTT Viability Assay Protocol
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The MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) viability assays were carried out as per manufacturers' instructions (Sigma). The L929 cells were plated at 50,000 cells per well in a 96-well flat-bottom plate (Becton-Dickinson Labware, Franklin Lakes, NJ). The cells were cultured with decreasing concentrations of the compounds (in duplicate), beginning at 0.50 mg/ml and decreasing by half over eleven additional dilutions. As controls, cells remained untreated (media alone) or were treated with equivalent dilutions of dimethylsulfoxide (DMSO, vehicle control) (Sigma). Following a 48 h incubation, the MTT reagent was added at 10% of the total volume per well. Following a 4 h incubation, the formed crystals were solubilized by removing approximately ¾ of the supernatant and adding 50 µl of solvent (10% Triton × and 1% 12 N HCl in isopropanol) (Sigma). All wells were vigorously pipetted (without forming bubbles) to help dissolve the crystals. The plates were then read in a 96-well plate spectrophotometer at 570 nm.
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