Cytosolic calcium imaging in olive pollen

Cytosolic Ca²⁺ levels were determined spectrofluorometrically using the probe FURA-2 AM (Del Pino et al., 2019a (link),b (link)). Olive pollen (100 mg) from sub-samples stored in the dark at 5°C was suspended in 10 ml PBS and hydrated for 2 days at 25°C. Hydrated pollens were harvested by centrifugation at 1,000 g × 4 min and then resuspended in 2 ml HBSS buffer (120 mM NaCl, 5.0 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 5 mM glucose, 25 mM Hepes, pH 7.4). Pollen suspensions were incubated in the dark with FURA-2 (2 μl of a 2-mM solution in DMSO) for 120 min, followed by centrifugation at 1,000 g × 4 min. Pollens were then harvested and suspended in ~10 ml of HBSS containing 0.1 mM EGTA, which was included to rule out or, at least, minimize a potential background due to contaminating ions (to obtain a suspension of 1 × 10⁶ of pollen granules hydrated per ml). Fluorescence was measured in a Perkin-Elmer LS 50 B spectrofluorometer (ex. 340 and 380 nm, em. 510 nm), set with a 10-nm and a 7.5-nm slit width in the excitation and emission windows, respectively. Fluorometric readings were taken after 300–350 s. When required, samples of pollen, H₂O₂ and Meg were added for specific purposes, as described in the Results section. Cytosolic calcium concentrations [(Ca²⁺)_c] were calculated as described in Grynkiewicz et al. (1985) (link).