All live cell images of the GFP-Rab5a cell line haven been taken with a Carl Zeiss Axio Imager A1 and imaging was performed as described [63 (link)]. A 100×/1.4–numerical aperture lens combined with a Hamamatsu Orca C4742-95 camera was used. Parasite cultures were stained with 1 μg/mL DAPI for 15 min before imaging. Exposure time was identical for all acquired images on the GFP channel. Parasites to be analysed were selected from DIC images. The corresponding GFP image were loaded into ImageJ (v1.51n) [61 (link)]. Setting to be recorded were max grey value and mean grey value. The background fluorescence of the images as well as the maximum fluorescent intensity of the brightest focus within the parasite and the background fluorescence of each parasite (excluding GFP foci) were measured. Image background fluorescence intensity was subtracted from the latter two values and the ratio was calculated. For plotting of data GraphPad Prism (V. 7.01) was used. Representative images were processed with Corel Photo-Paint x6 by adjusting brightness and intensity (V. 16.4.1.1281).
Orca c4742 95
Manufactured by Hamamatsu Photonics
The Orca C4742-95 is a scientific grade digital camera manufactured by Hamamatsu Photonics. It is designed for high-performance imaging applications. The camera features a CCD sensor and offers adjustable exposure times, resolution, and frame rates to suit various experimental requirements.
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Lab products found in correlation
3 protocols using orca c4742 95
1
Quantifying GFP-Rab5a Localization in Parasites
2
Fluorescence Microscopy of DAPI-Stained Parasites
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Fluorescence microscopy was carried out essentially as described (40 (link)). Briefly, 1 μg/μl DAPI was added to the parasites in culture medium and incubated for 15 min at room temperature. For imaging, a drop of this suspension was placed on a glass slide and covered with a coverslip. Images were acquired with a Zeiss AxioImager M1 or M2 microscope with a Hamamatsu Orca C4742–95 camera controlled by AxioVision software. A 63× plan-apochromate oil immersion objectives with an aperture of 1.4 was used for all images. Brightness and intensity of images adjustments and overlays were done using Corel Photo Paint (version X6). To assess knock sideways efficiency, a line crossing the nucleus was drawn through fluorescence images. The ‘plot profile’ function of ImageJ was then used to assess the fluorescence intensity of the DAPI signal and this was compared with the intensity profile of the same line in the GFP (TAF1/BDP5) signal.
3
Live Cell Imaging of GFP/mCherry-Expressing Parasites
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Live cell imaging of parasites endogenously expressing GFP and/or episomally expressing mCherry was carried out as previously described 64 (link) by placing the parasites on a glass slide and covering them with a cover slip. To visualise parasite nuclei, parasites were incubated with 1 mg/ml 4',6'-diamidine-2'-phenylindole dihydrochloride DAPI (Roche) or 50 ng/ml Hoechst 33342 (as indicated in the figure legends) in RPMI medium for 10 min at 37 C. For tubulin staining the parasites were harvested and resuspended in medium containing 1:1000 Tubulin Tracker TM Deep Red (Thermo Fisher Scientific; resolved in DMSO according to manufacturer's instructions). The cells were incubated for 20 minutes at 37 C in the shaking incubator (800 rpm) and 50 ng/ml Hoechst 33342 was added for additional staining of nuclei. The parasites were imaged with a Zeiss AxioImager M1 or M2 equipped with a Hamamatsu Orca C4742-95 camera and using either a 100x /1.4-numerical or a 63x /1.4-numerical aperture lens. Images were taken using the AxioVision software (version 4.7). The brightness and intensity of images were adjusted, and the channels were merged in Corel Photo-Paint (version X6). Corel Draw (version X6) was used for preparing the figures.
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