Annexin V-FITC Assay for Fungal Cell Death

Exposed phosphatidylserine in mASAL treated R. solani hyphae was detected using FITC-conjugated annexin V (Annexin-V FITC Apoptosis Kit, Sigma) as described by Madeo et al. [29 (link)] with some modifications. Both control (treated only with PBS) and mASAL treated (20 μg/ml for 48 h) fungal mycelia were harvested and washed with sorbitol buffer (1.2 M sorbitol, 0.5 mM MgCl₂, and 35 mM K₂HPO₄, pH 6.8). The cell walls were digested with 2 % Macerozyme R-10 (Sigma) and 15 U/ml lyticase (Sigma) in sorbitol buffer for approximately 3 h at 28 °C. The cells were harvested and washed with binding buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, and 2.5 mM CaCl₂) containing 1.2 M Sorbitol (binding-sorbitol buffer). To 96 μl hyphal suspensions in binding-sorbitol buffer, annexin V-FITC and PI are added to a final concentration of 1.2 μg/ml and 5 μg/ml respectively. The resulting suspension was then incubated at room temperature for 20–30 min. Following this the cells were immediately visualized using a confocal laser scanning microscope. A filter for FITC (excitation at 450–500 nm and emission at 515–565 nm) and PI (excitation at 550/25 nm and emission at 605/70 nm) was used. The experiments were performed in triplicate.