Development of multiparametric analysis of human PBMC by flow cytometry: Determination of inflammatory and calcic profile

Introduction Myocardial infarction affects more than 120,000 people every year in France. Despite the increasing effectiveness of patient management, the morbi-mortality of this condition remains high. Peripheral blood mononuclear cells (PBMC) have been recently shown to present a phenotype characteristic of a pathology, providing prognostic and/or diagnostic information. Objective We aim to develop a protocol for inflammatory and calcium phenotyping of human PBMC by flow cytometry. Methods After thawing PBMC from healthy patients, cell viability is followed by Trypan blue staining to define the optimal time for use. Regarding cell phenotyping, lymphocytes are Methods distinguished from monocytes according to their morphological criteria. Using the CD14, CD16, CCR2, and CX3CR1 markers, pro-inflammatory monocytes are further differentiated from anti-inflammatory monocytes. To analyze Ca2+ fluxes, different cell compartments are marked with chemical probes measuring the Ca2+ present in each compartment: FuraRedAM for cytosol, Rhod2AM for mitochondria, and MagFluo4AM for the reticulum. Dose-response study is performed to determine the optimal concentrations of each probe. Correct addressing of the probes in each compartment is verified by confocal microscopy. Using a pharmacological approach, the optimal conditions for measuring Ca2+ exchange between the different cell compartments will be determined. Results Cell viability remains stable during 8 hours but drops at 24 hours. Therefore, cytometer analyzes are carried out on the same day of thawing. Dose–response experiments and development of the multi-labeling protocol to phenotype the different PBMC populations are ongoing. Conclusion Once the protocol will be set up, PBMC from an existing cohort of post-myocardial infarction patients will be analyzed to better define the molecular signature of myocardial ischemia-reperfusion lesions by correlating the inflammatory/calcium PBMC profile with cytokines profile.