A methodology for detection of reactive oxygen species of neutrophils by by flow cytometry

Objective:To establish a method for detection of reactive oxygen species (ROS) of human polymorphonuclear cells (PMN) by flow cytometry.Methods:The whole fresh peripheral blood or Percoll separated PMN were incubated with dihydrogenrhodamine 123 (DHR123) or 2',7'-Dichlorodihydrofluorescin diacetate (DCFH-DA) as fluorescence probes for 15 min,followed adding PMA for 5-90 minutes at 37℃.The fluorescence intensities of rhodamine 123 and dichlorofluorecin were detected by flow cytometry,which reflecting the amount of reactive oxygen species within PMN.Results:By using DHR123 as a fluorescence probe,the tendency of the production of ROS generated in whole blood or separated PMN was similar when PMA incubated with them at 37℃,the amount of ROS were increased from 5 min to 60 min,reached a peak at 60 min,and decreased between 60 min to 90 min.By using DCFH-DA as fluorescent probe,the amount of ROS generated in whole blood stimulated by PMA reached the maximum in 5 min and thereafter decreased rapidly,whereas the production of ROS generated by isolated PMN increased depended on the time from 5 min to 30 min and reached platform between 60 min to 90 min;the RHO fluorescence intensity was apparently inhibited by formaldehyde-containing reagents.Conclusions:The application of flow cytometry to measure the reactive oxygen species within the neutrophils is simple,reliable and stable method.