Intracellular nitric oxide assessment in whole blood leukocytes by flow cytometry: Optimization and applicability to monitor patients with chronic graft nephropathy

Flow cytometry has been proposed as an alternative method for direct determination of intracellular NO by using the 4,5-diaminofluorescein-diacetate (DAF-2DA) as a fluorescent probe. In the present study, the protocol for intracellular NO determination in peripheral blood monocytes and neutrophils of by flow cytometry was optimized and applied to monitor chronic graft nephropathy patients. The optimize method consists to incubate plasma-free whole blood samples with DAF-2DA at 2.0 µM for 180 min at 37 °C to determine the percentage of DAF-2T+ monocytes and neutrophils. Distinct intracellular NO profiles in monocytes and neutrophils from chronic graft nephropathy patients as compared to the healthy individuals. Although the pre-incubation with LPS was able to trigger higher percentages of DAF-2T+ monocytes and neutrophils in both groups, our data demonstrated that LPS had a greater impact on monocytes as compared to neutrophils, selectively in the group of healthy individuals. Moreover, our findings demonstrated that LPS had lower impact on monocytes from chronic graft nephropathy as compared to healthy individuals. Supplementary analysis revealed that the LPS impact tends to be resorted in those patients with longer post-transplant time, as demonstrated by a significant positive correlation index. Furthermore, our results demonstrated that AG had lower inhibitory impact on neutrophils as compared to monocytes, selectively in the group of chronic graft nephropathy patients. Taken together, this study showed a new approach to monitor the immunological status of patients with chronic graft nephropathy opening new perspectives of research regarding the monocyte and neutrophil functions in patient undergoing immunosuppressive therapy.