Deltavision algorithms

Autophagy induction was visualized in MCF-7 cells which transiently transfected with GFP-LC3, and then placed on the microscope stage covered with a 37°C chamber in which a humidified premixed gas consisting of 5% CO₂ and 95% air was infused. After the treatment with tetrandrine (10 μM), cells with GFP-LC3 fluorescence puncta were observed using 60X Olympus PlanApoN 1.42 oil objectives at 512 nm emission. Both Differential interference contrast (DIC) and fluorescent images were acquired at 5-min intervals using high magnification widefield epifluorescence microscopy. Images were captured as serial Z-sections of 1.0 μm interval by a Photometrics CoolSNAP HQ2 CCD camera on the Olympus IX71-Applied Precision DeltaVision restoration microscope, and the epifluorescence images were numerically deconvolved using DeltaVision algorithms (Applied Precision, Inc.).

The detection of endogenous LC3 was conducted using immunofluorescence staining method as described below. In brief, HeLa cancer cells on cover slips were fixed with 4% paraformaldehyde (Sigma, 158127-3KG, USA) for 20 min at room temperature and then rinsed with PBS. Coverslips were immersed in methanol at room temperature for 2 min. After washing with PBS, the cells were then incubated with anti-LC3 (1:200) in TBST (100 mM Tris HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20 and 5% BSA) overnight at 4 °C. After washing with PBS, the cells were incubated with anti-mouse secondary antibody (TRITC) (1:200) in TBST containing 5% BSA at 37 °C for 1 h in the dark. The coverslips were then mounted with FluorSave™ mounting media (Calbiochem, 345789, USA). Samples were imaged by widefield epifluorescence microscopy using Photometrics CoolSNAP HQ2 CCD camera on the Olympus IX71-Applied Precision DeltaVision restoration microscope (Applied Precision Inc, USA). All fluorescence images were deconvolved using DeltaVision algorithms (Applied Precision, Inc). The percentage of cells with autophagic induction was calculated by the number of the cells with increased formation of punctate LC3 fluorescence dots (≥10 dots/cell) over the total number of immunofluorescence-positive cells in the same field. A minimum of 1000 cells from randomly selected fields were scored.