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

Abstract [Ca 2+ ] 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 (proTα) and its decapeptide proTα(100–109). Human leukocytes were activated with LPS, proTα or proTα(100–109), directly or after 24 h stimulation, while neutrophils were directly challenged. Cells were loaded with Fluo-4 and cytoplasmic Ca 2+ alterations were recorded by flow cytometry. Direct challenge with 20 μg/mL LPS induced a measurable [Ca 2+ ] i increase in macrophages and neutrophils. Monocytes and macrophages incubated for 24 h with LPS, proTα or proTα(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 Ca 2+ ion influx changes in immune cells. Their stimulation with proTα or proTα(100–109) generates an activating background, similar to LPS, allowing for the detection of [Ca 2+ ] i alterations induced upon subsequent challenge.