Eclipse ti s inverted

The astrocyte cultures were routinely imaged using phase-contrast microscopy techniques on a Nikon Eclipse Ti-S inverted microscope with Nikon Elements Basic Research software. For immunocytochemistry analysis, astrocyte cultures were fixed in 4% formaldehyde for 30 min, rinsed in phosphate-buffered saline (PBS) and permeabilized using 0.3% Triton X-100 plus 4% horse serum for 60 min. Primary antibodies were added (in PBS + 4% serum solution) at 4°C for 12 h. Primary antibodies to identify astrocytes and neurons were used. Rabbit anti-glial acidic fibrillary protein (GFAP; Millipore AB5804) was used to identify the intermediate filament protein present in astrocytes, and mouse anti-β-tubulin III (Sigma, C8198) was used to identify an element of microtubules expressed in neurons. After rinsing, Alexa 561 donkey anti-rabbit IgG and Alexa 488 donkey anti-mouse IgG secondary antibodies (1:500 in PBS + 4% serum) were added at 18–24°C for 2 h. Stretched or static astrocytic cultures were fluorescently imaged, using a laser-scanning confocal microscope (LSM 710 on an Axio Observer Z1; Zeiss, Oberkochen, Germany). For each culture, multiple confocal z-stacks were digitally captured and analysed.

MN9D cells grown in glass-bottomed plates were treated with 100 μM [1] for 12 h and then with 300 nM DAPI in KRB-HEPES for 15 min in the dark at 37 °C (26 (link), 41 (link)). Then cells were rinsed and KRB was added to cover the cell layer. Increase in nuclear DAPI fluorescence (Ex/Em at 358/461 nm) due to chromatin condensation was observed using a Nikon ECLIPSE Ti-S inverted fluorescence microscope. The controls were treated similarly except that the toxin was omitted from the initial incubation medium.

LNCaP cells were mixed in complete RPMI growth medium with 25% of non-conditioned or conditioned medium. Total of 150,000 cells in 2 ml of the mixture were added onto each well of the 6-well dish in triplicate. After 72 h, images were captured on a Nikon Eclipse Ti-S inverted microscope using a Ds-Fi1 camera (Nikon). Floating and adherent LNCaP cells were harvested and manually counted. For cell cycle analysis, cells were suspended in 1 ml of ice cold PBS and fixed by rapid addition of 3 ml cold absolute ethanol (Sigma). The cells were incubated for 1 h in 4 ml of 75% ethanol to complete fixation, washed 2 times in PBS, and then pelleted by centrifugation at 500×g for 7 min. Cells were incubated for 4–6 h at 4°C in 1 ml of PBS containing 30 U/ml RNase A (Qiagen) and 50 µg/ml propidium iodide. DNA content was measured with a flow cytometer and data analyzed using MultiCycle software (Phoenix Flow Systems). The viability of the LNCaP cells was determined as described for MSCs.