RPE PLK4 WT and ΔC cells were transfected with pGFP-LC3 (Addgene; plasmid #21073) using Mirius LT1 transfection reagent (Mirius Bio), plated on the cover slip of 35mm dishes (MatTek), and treated with 2 μg/mL doxycycline for 24 hours (if indicated). Live imaging of autophagosome traffic and autophagic flux was performed as previously described (27 (link)). Live, transfected cells were rapidly imaged (2000ms per Z-stack, 50 times) using: the Revolution XD spinning-disk microscopy system equipped with Yokogawa CSU-X1 confocal spinning disk head; Nikon Eclipse Ti inverted microscope surrounded by an Okolab cage incubator; iXon x3 897 EM-CCD camera; Andor laser combiner with four solid-state lasers at 405, 488, 561 and 640 nm and corresponding band-pass filter sets (Sutter); and ASI motorized stage with piezo-Z for rapid Z-stack acquisition as previously described (27 (link),28 (link)). Andor IQ2 software was used for image acquisition and Imaris X64 (Bitplane) for image analysis. Spots module was used to obtain speed and track displacement length of autophagosomes.
X3 897 em ccd camera
Manufactured by Oxford Instruments
The X3 897 EM-CCD camera is a high-performance scientific imaging device designed for low-light applications. It features a cooled, back-illuminated sensor and an electron-multiplying gain function to provide exceptional sensitivity and signal-to-noise ratio. The camera offers a range of user-configurable settings to optimize image quality and capture rate for specific experimental requirements.
Automatically generated - may contain errors
2 protocols using x3 897 em ccd camera
1
Live Imaging of Autophagic Flux
2
Characterization of ARPE-19 Cells on Laminin
Check if the same lab product or an alternative is used in the 5 most similar protocols
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ARPE-19 cells grown for 4 months on laminin-coated Transwell® filter inserts were blocked for 30 min with 1% bovine serum albumin (BSA) and stained for 1 h at room temperature with the following antibodies: rabbit anti-zonula occludens-1 (ZO-1, 1:200; Thermo Fisher Scientific, Waltham, MA, Cat # 61–7300), rabbit anti-claudin-2 (1:100; Thermo Fisher Scientific, Cat # 51–6100), and mouse anti-premelanosome protein (PMEL, 1:40; Thermo Fisher Scientific, Cat # MA5–13232). Antibodies were prepared in 1% BSA supplemented with 0.1% saponin. After three 5 min washes, the cells were stained with the actin stain rhodamine-phalloidin (1:200; Cytoskeleton, Denver, CO, Cat # PHDR1) and Alexa Fluor secondary antibodies (Thermo Fisher Scientific) at 1:500 in 1% BSA for 30 min. The cells were washed three times each for 5 min, stained with 4',6-diamidino-2-phenylindole (DAPI) for 5 min, rinsed, and mounted on glass slides. Images were captured using an Andor Revolution XD spinning disk confocal microscope using the 40X Plan Fluor oil objective (NA = 1.3, WD = 0.2 mm) and the iXon x3 897 EM-CCD camera. Images were processed using Imaris x64 software (Bitplane, South Windsor, CT).
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