Single and three‐color flow cytometry assay for intracellular zinc ion availability in human lymphocytes with Zinpyr‐1 and double immunofluorescence: Relationship with metallothioneins

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Abstract
Background: The amount of available intracellular zinc is pivotal to regulate many cellular processes, including oxidative stress response and apoptotic mechanisms. Therefore it is not surprising that zinc homeostasis and dyshomeostasis is involved in many physiological and pathological states, respectively. Cell permeable zinc probes allow intracellular applications with microscopy technology, but flow cytometry (FC) applications have been scarcely explored, albeit they can be suited to study zinc homeostasis in different cell types, including rare cells. Methods: We describe a FC method able to estimate intracellular zinc ion availability and the intracellular capability to activate a zinc signal after treatment with an NO‐donor (AcOM‐DEA/NO) in human PBMCs, using the fluorescent zinc‐specific probe, Zinpyr‐1 (ZP1), alone or in association with CD4‐PE and CD8‐Cychrome mAb. Results: This method was able to detect an increase/decrease of intracellular zinc available in human fresh cultured PBMC and in immune subsets using AcOM‐DEA/NO or TPEN, respectively. ZP1 mean fluorescence on gated histograms was sensitive to the amount of zinc added in the culture medium and significantly correlated to metallothioneins and total intracellular zinc. Conclusions: FC applications using ZP1 may be a fast and useful tool to study zinc homeostasis in immune cells. © 2006 International Society for Analytical Cytology