Quantitative analysis of viable cells was investigated using the Trypan blue exclusion method, which is based on the principle that the suspension of hCMEC/D3 cells is simply mixed with Trypan blue, then automatically visualized and counted by the Vi-Cell Analyzer using the Vi-Cell XR software (Beckman Coulter, Brea, CA, USA). In that protocol, only nonviable cells take up the dye. Before harvesting cells, the confluency level was visualized with an Olympus (Tokyo, Japan) 1X71 inverted phase-contrast microscope.
1x71 inverted phase contrast microscope
Manufactured by Olympus
Sourced in Japan
The 1X71 is an inverted phase-contrast microscope manufactured by Olympus. It is designed for observing specimens from below the stage. The microscope utilizes phase-contrast optics to enhance the visibility of transparent samples.
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2 protocols using 1x71 inverted phase contrast microscope
1
Trypan Blue Cell Viability Assay
2
Measuring Cancer Cell Transendothelial Migration
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ECIS arrays (8W10E+ , ibidi, Germany) were stabilised with l -cysteine (10 nM; 10 min incubation; Sigma, UK), washed in Hank’s balanced salt solution (Fisher, UK) and coated with 10 mg/mL fibronectin for 2 h followed by seeding of 7.5 × 104 cells/chamber of hCMEC/D3 in media supplemented with TNF-α (25 pg/mL). Resistance was monitored using an ECIS Zθ (Applied Biophysics, USA) system until a peak resistance was achieved. 2.0 × 104 of cancer cells were then added onto the endothelial cell monolayer. Resistance values were obtained in Ω. The confluency of the endothelial cell monolayer was also evaluated by using an Olympus 1X71 inverted phase contrast microscope.
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