4.5. Measurements of Cytosolic Ca2+

For cellular Ca²⁺ measurements, 3 × 10⁶ cells were incubated with 5 μM Fluo-4FF AM (Invitrogen, Carlsbad, CA, USA) in 5% CO₂ at 37 °C for 30 min, washed with Ca²⁺-Tyrode’s buffer, and resuspended in RPMI1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin. Before transfer to chamber slides, cells were washed, and resuspended in Ca²⁺-Tyrode’s buffer. For assays in Ca²⁺ free conditions, cells were incubated in with Na-Tyrode’s buffer without Ca²⁺ supplemented with 5 mM EGTA. Before microscopy, EGTA was removed by a washing step with Na-Tyrode’s buffer without EGTA. Sequential images were recorded with an LSM 880 system (Zeiss, Jena, Germany).