title: 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 24h 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 24h 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.
authors.affiliation: Department of Animal and Human Physiology, Faculty of Biology, University of Athens, Panepistimiopolis, Ilissia, 15784 Athens, Greece.
authors.forename: Nikos E
authors.initials: NE
authors.lastname: Papaioannou
authors.affiliation: Department of Animal and Human Physiology, Faculty of Biology, University of Athens, Panepistimiopolis, Ilissia, 15784 Athens, Greece; Cancer Immunology and Immunotherapy Center, Saint Savas Cancer Hospital, 171 Alexandras Avenue, 11522 Athens, Greece.
authors.forename: Ioannis F
authors.initials: IF
authors.lastname: Voutsas
authors.affiliation: Department of Animal and Human Physiology, Faculty of Biology, University of Athens, Panepistimiopolis, Ilissia, 15784 Athens, Greece.
authors.forename: Pinelopi
authors.initials: P
authors.lastname: Samara
authors.affiliation: Department of Animal and Human Physiology, Faculty of Biology, University of Athens, Panepistimiopolis, Ilissia, 15784 Athens, Greece. Electronic address: rtsitsil@biol.uoa.gr.
authors.forename: Ourania E
authors.initials: OE
authors.lastname: Tsitsilonis
mesh_terms: D000814:Aniline Compounds / Q000737:chemistry; D002118:Calcium / Q000032:analysis*; D002454:Cell Differentiation; D003584:Cytological Techniques / Q000379:methods*; D003593:Cytoplasm / Q000737:chemistry*; D003713:Dendritic Cells / Q000166:cytology / Q000276:immunology; D005434:Flow Cytometry; D006801:Humans; D007477:Ions / Q000032:analysis; D007962:Leukocytes / Q000187:drug effects / Q000276:immunology; D008264:Macrophages / Q000166:cytology / Q000276:immunology; D009000:Monocytes / Q000166:cytology / Q000276:immunology; D011498:Protein Precursors / Q000494:pharmacology*; D013947:Thymosin / Q000031:analogs & derivatives* / Q000494:pharmacology; D051197:Toll-Like Receptor 4 / Q000378:metabolism; D014966:Xanthenes / Q000737:chemistry
chemical_list: D000814:Aniline Compounds; C409648:Fluo 4; D007477:Ions; D011498:Protein Precursors; D051197:Toll-Like Receptor 4; D014966:Xanthenes; C040782:prothymosin alpha; D013947:Thymosin; D002118:Calcium
keywords: Calcium influx; Flow cytometry; Fluo-4; Human leukocytes; Prothymosin alpha; TLR4 endocytic pathway