P2X7 is an adenosine 5′-triphosphate (ATP)-gated ion channel. CD39 (ectonucleotidase triphosphate diphosphohydrolase-1) hydrolyses ATP to reduce its extracellular concentration to limit P2X7 activation. Both P2X7 and CD39 are present on leukocytes. Ficoll-Paque density gradient centrifugation is widely used to isolate human peripheral blood mononuclear cells (PBMCs), yet the extent to which other density gradient centrifugation methods influence the expression of cell surface molecules remains unclear. Using mass cytometry, this study compared the proportions of mononuclear leukocyte subsets and the relative amount of cell surface P2X7 and CD39 detected on these cells in paired, bar-coded cryopreserved human PBMC samples isolated by Ficoll-Paque or SepMate Tube density gradient centrifugation. Both techniques yielded similar proportions of mononuclear leukocyte subsets. P2X7 and CD39 were detected across all cell subsets, with the relative amount of P2X7 or CD39 comparable between separation methods. Relatively minor but statistically significant differences were observed for some populations. P2X7 expression was higher on CD3+, CD4+, and conventional CD4+ T cells, and naïve B cells, and lower on myeloid dendritic cells, while CD39 expression was lower on regulatory T cells in SepMate Tube samples compared to Ficoll-Paque samples. Overall, Ficoll-Paque and SepMate Tube density gradient centrifugation yield comparable results within PBMC samples, supporting the use of either method in studies examining immune phenotypes including the purinergic molecules P2X7 and CD39.
Elhage et al. (Mon,) studied this question.