A flow cytometric approach for studying alterations in the cytoplasmic concentration of calcium ions in immune cells following stimulation with thymic peptides

[Ca2+](i) alterations are vital in signaling pathways of cell activation. We tried to detect such changes, in intracellular signaling pathways downstream TLR4 in immune cells, following stimulation with prothymosin alpha (proT alpha) and its decapeptide proT alpha(100-109). Human leukocytes were activated with LPS, proT alpha or proT alpha(100-109), directly or after 24 h stimulation, while neutrophils were directly challenged. Cells were loaded with Fluo-4 and cytoplasmic Ca2+ alterations were recorded by flow cytometry. Direct challenge with 20 lg/mL LPS induced a measurable [Ca2+](i) increase in macrophages and neutrophils. Monocytes and macrophages incubated for 24 h with LPS, proT alpha or proT alpha(100-109) and challenged with LPS, displayed a robust response. Lymphocytes and iDCs exhibited no alterations. Conclusively, we assessed a flow cytometry-based method for monitoring Ca2+ ion influx changes in immune cells. Their stimulation with proT alpha or proT alpha(100-109) generates an activating background, similar to LPS, allowing for the detection of [Ca2+](i) alterations induced upon subsequent challenge. (C) 2016 Elsevier Inc. All rights reserved.