Eclipse te2000 u inverted phase contrast microscope

PC12 cells and/or primary neurons, seeded in a 96-well plate (1×10⁴ cells/well), were treated with different concentration of 6-OHDA (0-240 μM) for 24 h, with/without MPP⁺ (1 mM) or rotenone (1 μM) for 24, or with/without 6-OHDA (120 μM), MPP⁺ (1 mM) or rotenone (1 μM) for 24 h following pre-incubation with/without compound C (20 μM) for 2 h with 5 replicates of each treatment. In some cases, PC12 cells, infected with Ad-mTOR, Ad-S6K1-ca, Ad-myr-Akt, Ad-dn-AMPKα, or Ad-GFP (control), or infected with lentiviral shRNA to 4E-BP1 or GFP, respectively, were seeded at a density of 1 × 10⁴ cells/well in a 96-well plate or 5 × 10⁵ cells/well in a 6-well plate. Next day, cells were exposed to 6-OHDA (120 and/or 240 μM), MPP⁺ (1 mM) or rotenone (1 μM) for 24 h. Subsequently, cell viability was evaluated using an MTT assay by measuring the optical density at 570 nm under an ELx800 Microplate Reader (Bio-Tek Instruments, Inc. Winooski, VT, USA). The images for morphological analysis were taken with a Nikon Eclipse TE2000-U inverted phase-contrast microscope (Nikon, Tokyo, Japan) (200×) equipped with a digital camera.

Isolated murine primary neurons were treated with/without Cd (10 and 20 μM) for 24 h following pre-incubation with/without rapamycin (0.2 μg/ml) for 48 h with triplicates of each treatment. Subsequently, cells were fixed with 4% paraformaldehyde prepared in PBS for 2 h at 4°C. The cells were washed three times with PBS, and then stained with DAPI (4 μg/ml in deionized water) for 30 min at room temperature in the dark. Following a brief washing with PBS, slides were mounted in glycerol/PBS (1:1, v/v) containing 2.5% 1,4-diazabiclo-(2,2,2)octane. Photographs were taken with a fluorescence microscopy (Nikon 80i, Japan) equipped with digital camera. Cells with condensed nuclei were scored to be apoptotic. In addition, for cell morphological analysis, after treatment for 24 h, images were taken with a Nikon Eclipse TE2000-U inverted phase-contrast microscope (Nikon, Japan) (200×) equipped with digital camera.