Cellular endogenous NAD(P)H fluorescence as a label-free method for the identification of erythrocytes and reticulocytes

Reticulocytes and erythrocytes are the ultimate differentiated stages of erythropoiesis. In addition to being anucleate cells, they are characterized by the clearance of their mitochondrial pool or lack thereof. Given that for most research-oriented flow cytometry experiments erythrocytes and reticulocytes are often undesirable cell types, their identification and exclusion from analyses can be essential. Here, we describe a flow cytometric method based on cellular NAD(P)H-related autofluorescence, whose localization is mainly associated with mitochondria. By increasing the sensitivity of the specific NAD(P)H-fluorescence detector, we discovered a population with weak levels of NAD(P)H fluorescence signals whose immunophenotypical and physiological characterization in mouse bone marrow led to its identification as both erythrocytes and reticulocytes. Our method showed comparable sensitivity and specificity to the detection of red blood cells based on the absorption of light by oxyhemoglobin. This NAD(P)H-based approach consistently identified over 95% of the total pool of erythrocytes and reticulocytes in bone marrow samples and revealed robust as over 93% of these two erythropoietic subsets were identified in melanoma tumor samples with the same method. The measurement of cellular endogenous NAD(P)H fluorescence, therefore, offers a reliable and straightforward alternative to identify erythrocytes and reticulocytes without additional immunostaining or the need to modify the cytometer's optical configuration. © 2018 International Society for Advancement of Cytometry.