FL83B cells were seeded in coverslips placed in 12 well plates (150k cells/well). Then, 1.5uM of either Chol-NTC-siRNA or Chol-MCT1-siRNA at final concentration was added for 72 hours. To stain mitochondria membranes, cells were incubated with Mitotracker at 37C (Thermofisher, cat M7512) for 45 min in serum-free media. Then, cells were fixed with 4% paraformaldehyde at room temperature for 30 min. Fixed cells were blocked by fresh permeabilization buffer (0.5% Triton, 1% FBS in PBS) at room temperature for 30 min and incubated with 1:100 Anti-MCT1 (Proteintech, cat 20139-1-AP) overnight at 4C. As a secondary antibody, 1:1000 goat-anti-Rabbit-488 was used, while cells were protected from light. Coverslips were mounted on Prolong Gold antifade Mountant with Dapi (Invitrogen, cat P35934). Images were acquired using an Olympus IX81 microscope (Central Valley, PA) with dual Andor Zyla sCMOS 4.2 cameras (Belfast, UK). Images were quantified using ImageJ software.
Andor zyla scmos 4.2 cameras
Manufactured by Oxford Instruments
Sourced in United Kingdom
The Andor Zyla sCMOS 4.2 cameras are high-performance scientific CMOS (sCMOS) imaging sensors designed for a wide range of applications. The cameras feature a 4.2-megapixel sensor with a pixel size of 6.5 μm and offer a maximum frame rate of 100 frames per second. The Zyla sCMOS 4.2 cameras provide low noise, high dynamic range, and fast readout speeds, making them suitable for various scientific imaging techniques.
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2 protocols using andor zyla scmos 4.2 cameras
1
Quantifying Mitochondrial MCT1 Expression
2
Quantifying Mitochondrial Membrane Dynamics
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FL83B cells were seeded in coverslips placed in 12-well plates (150k cells/well). Then, 1.5 µM of either Chol-NTC-siRNA or Chol-MCT1-siRNA at final concentration was added for 72 hr. To stain mitochondrial membranes, cells were incubated with Mitotracker at 37°C (Thermo Fisher, cat M7512) for 45 min in serum-free media. Then, cells were fixed with 4% paraformaldehyde at room temperature for 30 min. Fixed cells were blocked by fresh permeabilization buffer (0.5% Triton, 1% FBS in PBS) at room temperature for 30 min and incubated with 1:100 Anti-MCT1 (Proteintech, cat 20139-1-AP) overnight at 4°C. As a secondary antibody, 1:1000 goat-anti-Rabbit-488 was used, while cells were protected from light. Coverslips were mounted on ProLong Gold Antifade Mountant with DAPI (Invitrogen, cat P35934). Images were acquired using an Olympus IX81 microscope (Central Valley, PA, USA) with dual Andor Zyla sCMOS 4.2 cameras (Belfast, UK). Images were quantified using ImageJ software.
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