an assay to monitor degranulation of azurophilic granules in neutrophils following activation

An Assay to Monitor Degranulation of Azurophilic Granules in Neutrophils following Activation

To begin, stimulate the mouse neutrophils in HBSS buffer containing 1 millimolar calcium chloride and magnesium chloride with or without fMLP or TNF-alpha for 10 minutes at 37 degrees Celsius.
Then, pellet the cells twice at 800 times g for 5 minutes. Collect 45 microliters of supernatant. Transfer the supernatant to the assay plate and run it in duplicates.
To detect the myeloperoxidase activity, add 80 microliters of 0.75 millimolar hydrogen peroxide solution, and 100 microliters of TMB solution to 20 microliters of supernatant. Then, incubate it at room temperature in the dark.&#160;
After 30 minutes, quench the reaction by adding 0.8 N hydrochloric acid. Read the absorbance on a PHERAstar plate reader at 450 nanometers. If necessary, generate a standard curve using recombinant myeloperoxidase to calculate the absolute amount of myeloperoxidase in the sample.

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Morphological and Compositional Analysis of Neutrophil Extracellular Traps Induced by Microbial and Chemical Stimuli

Neutrophils function as the first line of cellular defense in an innate immune response by employing diverse mechanisms, such as the formation of neutrophil extracellular traps (NETs). This study analyzes the morphological and compositional changes in NETs induced by microbial and chemical stimuli using standardized in vitro methodologies for NET induction and characterization with human cells. The procedures described here allow the analysis of NET morphology (lytic or non-lytic) and composition (DNA-protein structures and enzymatic activity), and the effect of soluble factors or cellular contact on such characteristics. Additionally, the techniques described here could be modified to evaluate the effect of exogenous soluble factors or cellular contact on NET composition.
The applied techniques include the purification of polymorphonuclear cells from human peripheral blood using a double density gradient (1.079-1.098 g/mL), guaranteeing optimal purity and viability (&#8805; 95%) as demonstrated by Wright's staining, trypan blue exclusion, and flow cytometry, including FSC versus SSC analysis and 7AAD staining. NET formation is induced with microbial (Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans) and chemical (phorbol myristate acetate, HOCl) stimuli, and the NETs are characterized by DNA-DAPI staining, immunostaining for the antimicrobial peptide cathelicidin (LL37), and quantification of enzymatic activity (neutrophil elastase, cathepsin G, and myeloperoxidase). The images are acquired through fluorescence microscopy and analyzed with ImageJ.

Presented here is a protocol for the induction and analysis of in vitro neutrophil extracellular traps (NETs). Quantification of DNA, cathelicidin (LL37), and enzyme activity yielded data that show the variability in the composition and morphology of NETs induced by microbial and chemical stimuli under similar controlled conditions.

Neutrophils function as the first line of cellular defense in an innate immune response by employing diverse mechanisms, such as the formation of ...

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Immunology and Infection

Quantifying Myeloperoxidase-DNA and Neutrophil Elastase-DNA Complexes from Neutrophil Extracellular Traps by Using a Modified Sandwich ELISA

Certain stimuli, such as microorganisms, cause neutrophils to release neutrophil extracellular traps (NETs), which are basically web-like structures composed of DNA with granule proteins, such as myeloperoxidase (MPO) and neutrophil elastase (NE), and cytoplasmic and cytoskeletal proteins. Although interest in NETs has increased recently, no sensitive, reliable assay method is available for measuring NETs in clinical settings. This article describes a modified sandwich enzyme-linked immunosorbent assay to quantitatively measure two components of circulating NETs, MPO-DNA and NE-DNA complexes, which are specific components of NETs and are released into the extracellular space as breakdown products of NETs. The assay uses specific monoclonal antibodies for MPO or NE as the capture antibodies and a DNA-specific detection antibody. MPO or NE binds to one site of the capture antibody during the initial incubation of samples containing MPO-DNA or NE-DNA complexes. This assay shows good linearity and high inter-assay and intra-assay precision. We used it in 16 patients with COVID-19 with accompanying acute respiratory distress syndrome and found that the plasma concentrations of MPO-DNA and NE-DNA were significantly higher than in the plasma obtained from healthy controls. This detection assay is a reliable, highly sensitive, and useful method for investigating the characteristics of NETs in human plasma and culture supernatants.

We present a protocol for a modified sandwich enzyme-linked immunosorbent assay technique to quantitatively measure two components of neutrophil extracellular trap remnants, myeloperoxidase conjugated-DNA and neutrophil elastase conjugated-DNA complexes,derived from activated neutrophils.

Certain stimuli, such as microorganisms, cause neutrophils to release neutrophil extracellular traps (NETs), which are basically web-like structures ...

Mast Cell Degranulation Assay to Study the Effect of a Target Chemical

To measure beta-hexosaminidase release, transfer 100 microliters of the cell suspension per well to a 96-well V-bottom well plate. Add 25 microliters of either stimulation or control solution to each well. Then, incubate the cells for 45 minutes at 37 degrees Celsius. Afterward, stop the reaction by placing the 96-well plate on ice for 5 minutes. Then, centrifuge the plate at 4 degrees Celsius for 4 minutes at 120 g.
Transfer 120 microliters of the supernatant to a flat-bottom 96-well plate, and place it on ice. Carefully avoid touching the cell pellets, but completely aspirate the supernatant. Next, add 125 microliters of lysis buffer to the cell pellets. Incubate the cell pellets for 5 minutes at room temperature, and resuspend them after the incubation by repeated pipetting.
Pipette 25 microliters of the 4-millimolar pNAG solution in the required wells of a new flat-bottom 96-well plate. Add 25 microliters of each supernatant, and 25 microliters of each cell lysate separately to the prepared pNAG solution. Incubate the reaction batches for 1 hour at 37 degrees Celsius. After the incubation, pipette 150 microliters of stop solution to each well to stop the reaction.
Next, analyze the plate sample light absorbance with a microplate reader at 405 nanometers with reference 630 nanometers for automatic background subtraction. In the acquisition program, check the box, &#34;Reference&#34;, and in the &#34;Absorbance&#34; program element menu, select &#34;630 nanometers&#34; for automatic background subtraction.

An Assay to Monitor Degranulation of Azurophilic Granules in Neutrophils following Activation

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