Av fitc pi

Uninfected and infected HKM (1 × 10⁶) were stained with AV-FITC-PI as described earlier and following manufacturer’s instructions (BD Pharmingen)^{15 (link)} The cells were observed under fluorescence microscope (Nikon Eclipse 400) within 30 min of adding the dye. Three fields with 100 cells each were studied to determine the percentage of apoptotic HKM^{15 (link)}.

For apoptosis studies the HKM (1×10⁶) were pre-treated separately with or without indicated concentrations of targeted or scrambled siRNAs, indicated concentrations of inhibitors for different time periods then infected with A. hydrophila at an MOI of 50 as described above. The HKM were subjected to Hoechst 33342 and Annexin-V-fluorescein isothiocyanate-Propidium Iodide (AV-FITC-PI, BD Pharmingen) staining 24 h p.i. as mentioned earlier [19] (link). For the Hoechst 33342 study, HKM were collected, washed and fixed with 3.7% paraformaldehyde solution at room temperature. After fixation, the HKM were stained with Hoechst 33342 (2 μg mL⁻¹ in 1×PBS) and visualized under fluorescence microscope (×40, Nikon Eclipse 400) within 30 mins of adding the stain. A total of 100 cells were studied in each field and three such fields were included to determine the percentage of apoptotic HKM. For the AV-FITC-PI study the HKM were collected, washed, fixed with 3.7% paraformaldehyde solution and stained with AV-FITC-PI following manufacturer's instructions. The HKM were observed under the fluorescence microscope (×40, Nikon Eclipse 400) within 30 mins of adding the dye. A total of 100 cells were studied in each field and three such fields were included to determine the percentage of apoptotic HKM.