Respiratory burst assay

Manufactured by Cayman Chemical

The Respiratory Burst Assay is a laboratory tool used to measure the production of reactive oxygen species (ROS) in cells, particularly in response to various stimuli. It provides a quantitative assessment of the respiratory burst, a process involved in the immune system's response to pathogens. The assay can be used to evaluate the functional activity of phagocytic cells, such as neutrophils and macrophages, and their ability to generate ROS as part of the host defense mechanism.

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Neutrophil/Monocyte Respiratory Burst Assay Kit (6 citations) Respiratory Burst Assay Kit (2 citations)

3 protocols using respiratory burst assay

Measuring Macrophage ROS Production

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ROS production by BMDMs (1X10⁵ events) was assessed using a commercially available kit (Respiratory Burst Assay, Cayman Chemical) according to the manufacturer’s protocol with the exception of stimulating the cells with PMA. Thus, incubating with dihydrorodamine 123 was an indicator of baseline ROS production, which was quantified by flow cytometry.

Owen A.M., Luan L., Burelbach K.R., McBride M.A., Stothers C.L., Boykin O.A., Sivanesam K., Schaedel J.F., Patil T.K., Wang J., Hernandez A., Patil N.K., Sherwood E.R, & Bohannon J.K. (2022). MyD88-dependent signaling drives toll-like receptor-induced trained immunity in macrophages. Frontiers in Immunology, 13, 1044662.

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Respiratory Burst Assay for BMDMs

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BMDMs were assessed using the Respiratory Burst Assay (Cayman Chemical Company, Ann Arbor, MI). In brief, BMDMs were incubated with dihydrorodamine-123, which fluoresces in the presence of reactive oxygen species (ROS). ROS were then elicited by incubation with 200nM phorbol 12-myristate 13-acetate (PMA) for 45 minutes. After stimulation, cells were washed with PBS and rhodamine mean fluorescence intensity was determined via flow cytometry.

Fensterheim B.A., Guo Y., Sherwood E.R, & Bohannon J.K. (2017). The Cytokine Response to LPS Does Not Predict the Host Response to Infection. Journal of immunology (Baltimore, Md. : 1950), 198(8), 3264-3273.

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Respiratory Burst Assay of Peritoneal Leukocytes

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Peritoneal leukocytes were assessed via the Respiratory Burst Assay (Cayman Chemical Company, Ann Arbor, MI). In brief, peritoneal leukocytes were obtained by peritoneal lavage with 2 ml cold PBS and incubated with dihydrorodamine-123. ROS were then elicited by 45 minutes of 200nM phorbol 12-myristate 13-acetate (PMA) stimulation. After stimulation, rhodamine 123 MFI was determined via flow cytometry. Neutrophils, macrophages, and monocytes were determined by FSC-A versus SSC-A.

Fensterheim B.A., Young J.D., Luan L., Kleinbard R.R., Stothers C.L., Patil N.K., McAtee-Pereira A.G., Guo Y., Trenary I., Hernandez A., Fults J.B., Williams D.L., Sherwood E.R, & Bohannon J.K. (2018). The TLR4 agonist monophosphoryl lipid A drives broad resistance to infection via dynamic reprogramming of macrophage metabolism. Journal of immunology (Baltimore, Md. : 1950), 200(11), 3777-3789.

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