Gal8-YFP-MDA-MB-231 cells were seeded at a density of 100,000 cells/well in 35-mm glass-bottom dishes (MatTek P35G-1.5-14-C) in DMEM containing 3% FBS and grown overnight. The next day, the cells were transfected with siRNA (10 nM final concentration) using RNAiMAX (Thermo Fisher, 7.5 μl/well in 1 ml of media). After 24 hours, the cells were washed twice with FluoroBrite DMEM (Thermo Fisher) and imaged immediately on a Leica DMi8 widefield fluorescence microscope. After image acquisition, images were exported in 16-bit TIFF format from the Leica LAS X software and processed in Fiji v1.53c to quantify the percentage of cells containing Gal8-YFP large foci, and the mean Gal8-YFP fluorescence intensity per cell [32 (link), 33 (link)].
Dmi8 widefield fluorescence microscope
Manufactured by Leica
Sourced in Germany
The Leica DMi8 is a widefield fluorescence microscope designed for high-performance imaging and analysis. It features an advanced optical system and a modular design, allowing for customization to meet the specific needs of various research applications.
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2 protocols using dmi8 widefield fluorescence microscope
1
Quantifying Gal8-YFP Foci in Cells
2
Microscopic Oxygen Imaging of Spheroids
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Before OC measurement, FGM was replaced with 1.5 ml of above-mentioned imaging medium. Measurements were performed using the STOp-Q method, as described previously (36 (link)), including the VisiSens TD Mic (PreSens Precision Sensing Technology GmbH, Regensburg, Germany) readout unit for microscopic O2 imaging. Ibidi dishes containing the spheroids embedded in type I collagen and NF8 cells were inserted into 6-well plates to fit into the imaging apparatus. Microscopic images over the entire area containing the spheroids and collagen/cell matrix were acquired using the tile scan function of the LASX software and a Leica DMi8 widefield fluorescence microscope (Leica, Wetzlar, Germany). Images were acquired with a 5x objective in the TXR channel (Excitation: 560/40, Emission: 639/75) and phase contrast. After a one-hour equilibration at 37°C in the incubator containing the STOp-Q device, measurements were performed in 10 second intervals for one hour using an exposure time of 150 ms (Figure 1
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