For live cell microscopy, cells were washed three times in PBS, resuspended in PBS with Hoechst 33342 (1 μg/ml) and then 5 μl of cell suspensions were placed on a glass slide. The cells were imaged using either a Leica DM5500B microscope with 100x objective and Neo 5.5 sCMOS camera or a Zeiss ImagerZ2 microscope with 63x or 100x objective and Hamamatsu Flash 4 camera. For cell swimming analysis, a 61 s video of 512 frames under darkfield illumination was captured using a 10x objective. Particle tracks were traced and quantified (mean speed and cell directionality (ratio of velocity to speed) automatically as previously described [40 (link)]. Individual tracks were extracted after manual inspection for presence of 1 flagellum or cells connected via their flagella, with the track statistics then plotted as for the entire population.
Dm5500b microscope
Manufactured by Zeiss
The DM5500B is a high-performance microscope system designed for advanced imaging and analysis applications. It features a modular design, allowing for customization to meet specific research or analytical needs. The DM5500B provides excellent optical performance, advanced imaging capabilities, and reliable operation.
Automatically generated - may contain errors
2 protocols using dm5500b microscope
1
Live Cell Microscopy and Swimming Analysis
2
Visualizing C. elegans Germline Development
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The procedure for dissection, immunostaining, and imaging of germlines was as described in Day et al. (2018) (link). For immunostaining embryos, worms were dissected at the vulva to release embryos. The primary and secondary antibodies and their dilution factors used for immunostaining 3xFLAG-, GFP- and OLLAS-tagged proteins, and endogenous P granule proteins are described in Supplemental Table 4. The images were acquired with a Leica DFC300G camera attached to a Leica DM5500B microscope or with a Zeiss LSM 880 confocal microscope, stitched together using FIJI/ImageJ (if needed), and cropped using Adobe Photoshop CS3. Colocalization analysis was performed on confocal images in FIJI/ImageJ using the Just Another Colocalization Plugin (JACoP; Bolte and Cordelières, 2006 (link)). In Figure 5 , P granules were imaged with the same laser power/gain for panels A, B, and D (early and dispersed P granules) but with a lower laser power for panel C (wild-type late-embryo P granules) to avoid overexposure.
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