Calcium Mobilization and CD5 Expression Analysis

Calcium mobilization was also measured using flow cytometry and the high affinity calcium indicator Fluo-4 (ex:470–490 nm and em: 520–540 nm). Cells were surface stained with an anti-CD4⁺-APC antibody (17–0041; eBioscience). T cells were loaded for 30 mins as previously published with pluronic acid and 1mM Fluo-4-acetoxymethyl ester (Invitrogen) in Ringer solution (150 mM NaCl, 10 mM glucose, 5 mM of HEPES, 5 mM of KCl, 1 mM MgCl₂, and 2 mM CaCl₂, pH 7.4) [35 (link)]. Intracellular calcium mobilization was initiated by adding 50 ng/ml of PMA (phorbol 12-myristate 13-acetate) and 1 μg/mml of ionomycin [36 (link)]. For further analysis done in FlowJo, the lymphocyte population was gated in a forward and side scatter gate and singlets. From this gate a second gate was created specific for CD4⁺ T cells [37 (link)]. Intracellular calcium flux was measured in the CD4⁺ T cell gate using the FlowJo kinetics tool.
For CD5 expression analysis, spleen single cell suspensions from naïve and stimulated (day 3 and day 8) were stained with anti-CD5-PE (12–0051; eBioscience), and anti-CD4-APC (17–0041; eBioscience) and analyzed on the flow cytometer (BD Accurri C6).