Neutrophil ROS production was measured through the oxidization of non-fluorescent dihydrorhodamine 123 to fluorescent rhodamine 123 by ROS using the Neutrophil/Monocyte Respiratory Burst Assay Kit (Catalogue number 601130, Cayman Chemicals, Ann Arbor, MI) according to manufacturer’s instructions [24 (link)]. In brief, 100 μl EDTA–anticoagulated whole blood and 10 μl of 5 μg/ml dihydrorhodamine 123 working solution were added to each 5-ml round-bottom FACS tube (Catalogue number 352054, Corning), mixed well, and incubated in a 37°C water bath for 15 min. To stimulate ROS production, phorbol 12-myristate 13-acetate (PMA; Catalogue number 400145, Cayman Chemicals) in dimethyl sulfoxide (DMSO) was added to each cell suspension to reach a final concentration of 200 nM or as indicated in the dose-response assay, and the same volume of PBS was added to controls. After incubation in a 37° C water bath for 45 min, 3 ml PBS was added to each tube followed by centrifugation of the cells (700 × g for 5 minutes at room temperature). Cell pellets were resuspended in 200 μl PBS for immunostaining (CD66abce-PE) and live/dead detection via flow cytometry as described above for neutrophil phagocytosis. ROS production was measured by detection of rhodamine 123 signal in the FITC channel of live CD66abce+ cells.
Neutrophil monocyte respiratory burst assay kit
Manufactured by Cayman Chemical
The Neutrophil/Monocyte Respiratory Burst Assay Kit is a lab equipment product designed to measure the respiratory burst activity of neutrophils and monocytes. It provides a quantitative assessment of the oxidative burst response in these immune cells.
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Lab products found in correlation
Round bottom facs tube, by Corning (1 mentions)
Brilliant violet 510 conjugated anti mouse ly6g 1a8, by BioLegend (1 mentions)
Facscanto 2, by BD (1 mentions)
Cd45 vf450, by Cytek Biosciences (1 mentions)
Ghost dye 510, by Cytek Biosciences (1 mentions)
Cd11b apc cy7, by Cytek Biosciences (1 mentions)
6 protocols using neutrophil monocyte respiratory burst assay kit
1
Neutrophil Respiratory Burst Assay for ROS Detection
2
Evaluating Neutrophil Respiratory Burst
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To evaluate bactericidal activity of neutrophils, ROS production in response to PMA was evaluated using the Neutrophil/Monocyte Respiratory Burst Assay Kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer’s protocol. Peripheral blood was incubated with PE-conjugated anti-Ly6G mAbs for 15 min on ice and then labeled with dihydrorhodamine 123 (DHR 123) for 15 min at 37°C. Next, cells were stimulated with 0.2 μM PMA for 45 min at 37°C. DHR123 signals in the neutrophils were evaluated using a flow cytometer.
3
Neutrophil Respiratory Burst Capacity Assay
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Neutrophils isolated as described above were then counted and diluted to a concentration of 1x10^6 cells/ml. Cells were stained with surface markers mentioned above (CD45, CD11b, Ly6G, Ly6C). Following surface staining, dihydrorhodamine 1,2,3 dye (DHR), PMA and incubation buffer were used according to manufacturer’s instructions (Neutrophil Monocyte Respiratory Burst Assay Kit, Cayman Chemical). 100ul of cells were taken and incubated with DHR and various concentrations of PMA (0, 25, 50, 100, 200, 400 nM PMA) for 45 minutes at 37°C. Flow cytometry was used to identify neutrophils, and measure median fluorescence intensity of the DHR signaling per cell.
4
Respiratory Burst Assay in HUVEC-PMN Coculture
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HUVEC cells were seeded into a 24-well plate at a concentration of 1x105 cells/well. After 24 h, the supernatants were discarded and 5x105 human PMNs were added, followed by PTC, CRT0066101 (5 µM) or DMSO treatment. This coculture was incubated for 3 h 45 min at which point fMLP was added (100 nM). After 15 min the supernatants were collected, centrifuged for 2 min at 2000 rpm at RT and stored at -20°C. Respiratory burst experiments with PMNs were performed using the Neutrophil/Monocyte Respiratory burst assay kit (#601130, Cayman Chemical) according to the manufacturer´s instructions. PMNs were incubated with DHR 123 reagent at a concentration of 1x105 cells/mL in Assay Buffer (RPMI, 1 mM Calcium Chloride) for 30 min before they were added to HUVECs that had been seeded into a 24-well plate at a concentration of 1x105 cells/well and grown for 24 hours. This coculture was incubated for 3 h 45 min at which time point the PMNs were harvested. Next, fMLP was added (100 nM) to the harvested PMNs and incubated for 15 min. Subsequently, PMNs were resuspended in FACS buffer, stored on ice and analyzed by flow cytometry using the FITC-A channel.
5
Neutrophil Respiratory Burst Assay
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Respiratory burst in isolated neutrophils was assessed using the Neutrophil/Monocyte Respiratory Burst Assay Kit (Cayman Chemical, Ann Arbor, Michigan). Following NA treatment, the cells were washed and resuspended in RPMI supplemented with 50 μl of 1 M calcium and 2% fetal calf serum. Cells were then seeded into a 96‐well V‐bottom plate at 2 × 106 cells/ml and incubated with 50 ng of dihydrorhodamine (DHR) 123 for 15 min in a 37°C, 5% CO2‐supplemented incubator. Two hundred nanomolal phorbol myristate acetate (PMA) assay reagent was then added to each well and incubated for 30 min in a 37°C, 5% CO2‐supplemented incubator. Following incubation, the cells were immediately placed on ice before being washed and resuspended in respiratory burst assay buffer. Cells were then stained with Brilliant Violet 510‐conjugated anti‐mouse Ly6G (1A8, BioLegend) and FITC‐conjugated anti‐mouse CD11b (M1/70, BioLegend). A BD FACSCanto II (BD Biosciences) flow cytometer was used to examine the fluorescence of cells. FlowJo software was used to gate the neutrophil cell population, determine the median fluorescence intensity (MFI) of DHR 123 for this population, and examine the expression of CD11b on neutrophils.
6
Profiling Immune Cell Functionality
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Single-cell brain and bone marrow suspensions were washed twice in 1X sterile, cold PBS, followed by viability staining with Ghost Dye 510 (Tonbo Biosciences) for 30 minutes in the dark at room temperature. Cells were centrifuged at 500g for 5 minutes at 4°C, the supernatant was discarded and the pelleted cells were re-suspended in 100ul of 1:100 FC Receptor Block (Tonbo Biosciences) for 10 minutes at room temperature. Cells were then stained with following surface antibodies: CD45-vf450, CD11b-APC-Cy7, Ly6G-Pe-Cy7, Ly6C-APC (Tonbo Biosciences) for 30 minutes at room temperature, protected from light. Following surface staining, samples were incubated with dihydrorhodamine 1,2,3 (DHR) and 200 nM PMA for 45 minutes at 37°C according to the manufacturer’s instructions (Neutrophil Monocyte Respiratory Burst Assay Kit, Cayman Chemical), then washed and analyzed immediately for rhodamine fluorescence on a Beckmann Coulter Cytoflex S Flow Cytometer. Data was analyzed using FlowJo (TreeStar).
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