Air laser scanning confocal microscope system

The confocal microscopy was done using previously followed method with slight modification (Bhushan et al. 2007 (link)). The PC-3 cells (5 × 10⁵ cells/well) were seeded in 6-well plate and allowed to adhere for 24 h. The cells were treated with IC₅₀ and IC₇₀ concentration of CoCl₂·H₂O for 12–14 h. Thereafter, the cells were washed with phosphate buffer saline and incubated with 4 % PFA for half an hour. After fixation, the cells were stained with 4,6-diaminidino-2-phenylindole (DAPI), and slides were prepared for imaging with confocal microscope. The slides were scanned under Nikon eclipse TiE inverted fluorescence microscope equipped with a Nikon AiR laser scanning confocal microscope system (Nikon Corporation, Japan). Fluorescence was observed with long pass 488 emission filters.

Apoptosis induced by treatments of CoCl₂·6H₂O in PC-3 cells was analysed using Annexin V-FITC detection kit (Sigma Aldrich Inc., USA.). The procedure was followed as given in the kit. The cells were viewed under Nikon eclipse TiE inverted fluorescence microscope equipped with a Nikon AiR laser scanning confocal microscope system (Nikon Corporation, Japan).