The phagocytosis of PMNs plays an important role in the process of resisting bacterial, fungal and other pathogenic microorganisms infection. The process of phagocytosis consists of several major stages: chemotaxis, adhesion, endocytosis, intracellular oxygen-dependent (respiratory burst) and oxygen-independent killing. Under normal conditions, PMNs are activated after bacterial infection, and the enhancement of PMNs phagocytosis and oxidation is an important innate immune defense mechanism of the body. Therefore, by observing the activation status of PMNs, we can help to understand the bacterial infection status of the organism. PMNS function was detected by flow cytometry (FMC), PMA was used as high performance stimulant, and dihydroardamine was used as fluorescence substrate. When neutrophils are stimulated, they produce reactive oxides, and rhodamine dihydrogen oxide is rhodamine 123, which can emit fluorescence. This reaction can be terminated by hemolysin.The reactive oxides produced can be expressed in terms of fluorescence intensity.PMNS activation and the body's innate immune capacity.It can be used to study the functional changes of neutrophils in various pathological conditions, such as infection, autoimmune disease, emergency, tumor, surgery, etc., and to evaluate the effect of drugs.The kit can be used not only in humans, but also in mice, rats, rabbits, dogs, cattle and other species.
PMA solution dihydrorhodamine hemolysin
1. Sterile heparin anticoagulant whole blood 50ul, adding stimulant (such as PMA) 50ul, 37°C Incubate for 15 minutes.
2.Add dihydrorhodamine 25ul, 37°C Incubate in dark for 5 minutes.
3.Dilute the hemolysin with sterile deionized water at 1:10, add 1ml of the diluted hemolysin, and hemolysis at room temperature without light for 15 min.
4. Wash the hemolysin with PBS twice, centrifuge at 1500rpm for 5 min, and remove the supernatant.
5.The cells were resuspended with 0.5mlPBS and detected by flow cytometry.
1 Sawyer, D.W., Donowitz, G.R. & G.L. Mandell. 1989. Polymorphonuclear neutrophils: An effective antimicrobial force. Rev. Infect. Dis. 11: S1532-S1544.
2．Richardson MP, Ayliffe MJ, Helbert M, et al. A simple flow cytometry assay using dihydrorhodamine for the measurement of the neutrophil respiratory burst in whole blood: comparison with the quantitative nitrobluetetrazolium test. J Immunol Methods,1998,219(1-2):187-193.
3．Smith, R.M. & J.T. Curnutte. 1991. Molecular basis of chronic granulomatous disease. Blood 77: 673 -686.
4．Jirapongsananuruk O, Malech HL, Kuhns DB, et al. Diagnostic paradigm for evaluation of male patients with chronic granulomatous disease, based on the dihydrorhodamine 123 assay. J Allergy Clin Immunol. 2003,111(2):374-9.
5．Walrand S, Valeix S, Rodriguez C, et al. Flow cytometry study of polymorphonuclear neutrophil
oxidative burst: a comparison of three fluorescent probes. Clin Chim Acta, 2003, 331(1-2):103-110.
6．Rothe G, Oser A & G. Valet. 1988. Dihydrorhodamin 123: a new flow cytometric indicator for respiratory burst activity in neutrophil granulocytes. Naturwissenschaften 75: 354 -355.
|Forward Angle and lateral Angle dot map|
|Static, activated neutrophil FL1 fluorescent column map|
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