Flow cytometric calcium flux assay: Evaluation of cytoplasmic calcium kinetics in whole blood leukocytes

Abstract

In leukocytes, as in many other cell types, cytoplasmic calcium ([Ca 2+] i) changes play a key role in a series of pathways leading to activation. Here we describe a flow cytometric method allowing the simultaneous kinetic analysis of changes in [Ca 2+] i in the three types of leukocytes, i.e. monocytes, granulocytes and lymphocytes. Heparinised whole blood was diluted in phosphate buffered saline with Ca 2+ and 1 mM sodium pyruvate and incubated with the Ca 2+ indicator fluo3-acetoxymethyl ester. Leukocytes were identified by labelling with the phycoerythrin-conjugated antibody against CD45, the leukocyte common antigen. Resuspension of the cells in PBS with or without Ca 2+ allowed us to detect the origin of Ca 2+ changes. During flow cytometric analysis only CD45-positive cells were counted and monocytes, granulocytes and lymphocytes were evaluated separately. Baseline fluorescence of the fluo3–Ca 2+-complex was determined and changes in [Ca 2+] i after stimulation with the calcium ionophore A23187 or the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) were recorded over a time period of 150 s. Stimulation with A23187 resulted in a rise in [Ca 2+] i in all three leukocyte subpopulations. This rise was sustained in the presence of extracellular Ca 2+ (Ca 2+ ex) but had a transient character in the absence of Ca 2+ ex. For fMLP, [Ca 2+] i changes occurred only in monocytes and granulocytes and were transient irrespective of the presence or absence of Ca 2+ ex. In conclusion, the present method is a simple, fast and easy tool to analyse in vitro [Ca 2+] i changes over time in leukocytes under physiologically relevant conditions, without the need for their isolation or the lysis of erythrocytes. The whole blood approach allows a continuous interaction between the different leukocyte subpopulations and other blood components and a minimum of preparative manipulations avoids artefactual activation of the cells. A distinction can be made between Ca 2+ release from the intracellular stores and the entry of Ca 2+ from outside the cell. The approach allows to evaluate the effect of various agonists on [Ca 2+] i changes in leukocytes, with physiological, patho-physiological or therapeutic purposes.