Analysis of cell surface and intranuclear markers on non-stimulated human PBMC using mass cytometry.

Mass cytometry is a powerful tool that allows simultaneous analysis of more than 37 markers at the single cell level. Mass cytometry is of particular interest in the identification of a wide variety of cell phenotypes in autoimmune diseases. Moreover, cells can be labelled with palladium isotopes and pooled before staining (barcoding). Nevertheless, immunologists often face an important problem concerning the choice of markers to be included in a panel. This problem arises due to the incompatibility of different buffers used for the fixation and permeabilization of cells with various cell surface epitopes. In this study, we used a panel of 27 markers (19 surface markers and 8 intranuclear markers) to demonstrate disparities in the detection of cell surface antigens when comparing different buffers to stain unstimulated peripheral blood mononuclear cells. These disparities range from mild differences to very important differences in population frequencies depending on the buffers. Finally, we demonstrate the harmful effects of permeabilization prior to barcoding on the detection of some cell surface antigens. Here, we optimize a protocol that is suitable to use when targeting a large panel including both cell surface and intranuclear markers on unstimulated human peripheral blood mononuclear cells.