The C1C7 cells were seeded in a 96-well plate for 24 h in DMEM supplemented with 10% FBS, then loaded with the intracellular Na+ -fluorescent dye ION NaTRIUM Green-AM. After washing with DMEM supplemented with 10% FBS, cells were treated with Monensin (10 µM) in presence of the cell-impermeant TO-PRO™−3 Iodide dye (1 µM) to selectively stain nuclei of dead cells. The cells were incubated in controlled atmosphere (5% CO2; 37 °C) for 20 h in DMEM ( + Na+) or in DMEM (-Na+) in the cage incubator of the THUNDER Imager 3D Live Cell ( Leica Microsystems; Germany). The Na+ intracellular variations and appearance of cell death were recorded every 30 min by employing a semi-automated recording/analysis protocol of Thunder live-cell imager’ platform. Negative controls were performed using samples loaded with the singular dyes or without dyes. Changes of fluorescence were analyzed using ImageJ software v 1.53e.
Thunder imager 3d live cell
Manufactured by Leica
Sourced in Germany
The THUNDER Imager 3D Live Cell is a high-performance microscope system designed for live-cell imaging. It provides 3D imaging capabilities with fast acquisition speeds and advanced optics, enabling the observation and analysis of dynamic cellular processes in real-time.
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Lab products found in correlation
2 protocols using thunder imager 3d live cell
1
Monitoring Intracellular Sodium and Cell Death
2
Yeast Orf9b Colocalization and Mitochondrial Analysis
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Yeast strains were cultured in synthetic media to mid-log phase. Orf9b expression was induced by the addition of 0.5% galactose to the cultures. Cells were harvested after 4 h of galactose induction and images were taken using the Leica-Thunder imager 3D live cell using 100× oil objective (Leica Microsystems 0.55 S28 LEICA; Inverse, DMi 8 HC; PL Apo 100×/1.44 Oil CDRR CS, LAS X). Colocalization analysis of the Su9-mNeonGreen (green) signal and Orf9b-mScarlet (red) signal was performed using the ImageJ/Fiji Coloc2 plugin (Schindelin et al., 2012 (link)). Regions of interest (ROIs) outlining the cells were selected, followed by measurement of Pearson’s correlation coefficient in the selected ROIs. For the mitochondrial area analysis, an ImageJ/Fiji macro based on thresholding was created. ROIs defining the cells were selected manually for each image and the area was calculated using the analyze particles function in ImageJ/Fiji. Correlation and area results were plotted in RStudio using the Beeswarm package. Statistical analysis was performed in RStudio using the Kruskal–Wallis rank sum test followed by Dunn’s test adjusted with the Benjamini–Hochberg method.
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