Bone marrow neutrophils (2 × 105 cells) were seeded in Costar 96-well black plates (Corning, MA) in the presence of 0.5% fetal bovine serum and Sytox Green (5 μM), a cell impermeable DNA binding dye. Platelets were purified from whole blood collected in sodium citrate anticoagulant tubes and platelet-rich plasma was obtained by centrifugation. The cells were incubated with human resistin or PMA in the presence of platelets (106 cells) for indicated time at 37°C and then free DNA in culture medium was measured using time dependent fluorescence of Sytox Green probe (Fluostar OPTIMA spectrophotometer, (BMG LABTECH Microplate Readers, Alexandria, VA, USA), at an excitation wavelength of 492 nm and an emission wavelength 530 nm. To measure release of DNA in the lung of mice, BAL fluid (50 μl) was incubated with 50 μl of Sytox Green (5 μM) for 10 minutes followed by reading Sytox Green fluorescence.
Costar 96 well black plates
Manufactured by Corning
Sourced in Switzerland, United States
The Costar 96-well black plates are a type of laboratory equipment used for various scientific applications. These plates feature 96 individual wells arranged in a 8x12 grid pattern and are made of a black-colored material. The core function of these plates is to provide a platform for conducting assays, experiments, or other analytical procedures that require a multi-well format.
Automatically generated - may contain errors
Lab products found in correlation
Sytox blue, by Thermo Fisher Scientific (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Varioskan, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Roche (1 mentions)
Plasmin, by Merck Group (1 mentions)
Heparin, by Thermo Fisher Scientific (1 mentions)
Fluostar omega, by BMG Labtech (1 mentions)
Trypsin, by Promega (1 mentions)
Tristar multimode microplate reader lb941, by Berthold Technologies (1 mentions)
4 protocols using costar 96 well black plates
1
Quantifying Neutrophil DNA Release
2
Quantification of Neutrophil Extracellular Traps
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Neutrophils were incubated for 30 min in 50% RPMI/7% FCS and 50% BAL fluid or saline. When indicated, BAL fluids were neutralised beforehand with an anti-HMGB1 mAb (IBL International GmbH) or isotype control for 2 h. Neutrophils were then labelled with 5 µmol•L -1 Sytox blue (Invitrogen, Carlsbad, CA, USA) in RPMI/0.5% FCS with or without DNase I (200 IU•mL -1 ; Roche, Basel, Switzerland), seeded in Costar 96-well black plates (Corning Costar Corporation, Cambridge, MA, USA) and stimulated or not with 10 µmol•L -1 phorbol myristate acetate (PMA; Sigma-Aldrich) for 3 h at 37°C. The release of NETs (termed NETosis) was quantified by measuring fluorescence with a microplate fluorescence reader (Varioskan, ThermoFisher Scientific, Waltham, MA, USA).
3
TTR Amyloid Formation Kinetics
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Recombinant S52P or L111M TTR, 100 μL volumes at 1 mg/mL in PBS, pH 7.4 containing 10 μM ThT (Sigma‐Aldrich) in PBS and 12 μM heparin (Alfa‐Aesar) was incubated at 37°C in Costar 96‐well black plates (Corning Incorporated) in the presence of a protease/substrate ratio of 5 ng/μL trypsin (Promega, V5280) and 20 ng/μL plasmin (Sigma‐Aldrich, P1867) to yield a final enzyme:TTR ratio of 1:200 and 1:50, respectively. Control experiments with TTR variants incubated without enzyme were carried out in parallel. The plate was sealed with clear sealing film and subjected to 900 rpm double‐orbital shaking until the ThT signal reached a plateau. Bottom fluorescence was recorded at 500 s intervals (BMG LABTECH FLUOstar Omega). A two‐way ANOVA test was performed using GraphPad Prism 9 for pairwise multiple comparisons.
4
Quantifying Neutrophil Extracellular Traps (NETs)
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NETs formation was quantified using PicoGreen as described previously [27 (link)]. Lambda DNA of known concentration was serially diluted with Tris-EDTA (TE) buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) to create standard DNA samples. To follow NETs formation, 100 μl fresh neutrophils (1 × 105cells) were seeded in Costar 96-well black plates (Corning, Tewksbury, MA, USA) in the presence of 0.5 % FBS. Neutrophils were pretreated with buffer or 10 ng/ml HMGB1 for 30 min followed by stimulation with MPO-ANCA-positive IgG, or PR3-ANCA-positive IgG, or normal IgGs in an incubator containing 5 % CO2 at 37 °C for 3 h, respectively. For assay of the role of candidate receptors, certain groups of neutrophils were preincubated with relevant reagents for 30 min on ice before being pretreated with HMGB1. Some 100 μl of the PicoGreen dye diluted 1:200 in TE buffer was added to the microplate in order to make a final volume of 200 μl per well. Following incubation in the dark for 5 min at room temperature, the fluorescent signal of the sample was measured using the microplate fluorescence reader (TriStar Multimode Microplate Reader LB941, Berthold Technologies, Bad Wildbad, Germany), at an excitation wavelength of 480 nm and an emission wavelength of 530 nm.
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