Cells were resuspended in culture media and concentrated by cytospin centrifugation at 700g for 5 min onto SuperFrostPlus slides (ThermoFisher Scientific) using a Shandon Cytospin 3 cytocentrifuge. Slides were fixed for 3 min in cold methanol and stained with May-Grünwald-Giemsa (Sigma). Images were captured using a Leica DM5000b microscope in conjunction with a × 63 oil-immersion lens and an Olympus DP72 camera.
Dm5000b microscope
Manufactured by Olympus
The Olympus DM5000b is a high-performance microscope designed for advanced research and analysis. It features a robust optical system and a range of advanced functionalities to support a variety of imaging techniques. The DM5000b is capable of delivering high-quality images and data, making it a reliable tool for researchers and scientists.
Automatically generated - may contain errors
Lab products found in correlation
May gr nwald giemsa, by Merck Group (2 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (2 mentions)
Fluoview fv1000 confocal microscope, by Olympus (1 mentions)
Vs110 slide scanner, by Olympus (1 mentions)
Sp8 confocal system, by Leica (1 mentions)
Dm5000b microscope, by Leica (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
4 protocols using dm5000b microscope
1
Cytospin-based Cell Staining Protocol
2
Cytospin-based Cell Concentration and Staining
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Sorted transgene-positive or gated wild-type cells were concentrated by cytocentrifugation at 7 × g for 5 min onto SuperFrostPlus slides using a Shandon Cytospin 3 cytocentrifuge. Slides were fixed for 3 min in −20 °C methanol and stained with May–Grünwald Giemsa (Sigma). Images were captured using a Leica DM5000b microscope in conjunction with a ×63 oil-immersion lens and an Olympus DP72 camera.
3
Visualizing Candida-Induced Macrophage Extracellular Traps
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J774A.1 macrophage-like cells were infected with C. albicans at a MOI of 25:1 and incubated at 37°C in 5% CO2 for 1 h. Cells were then fixed to coverslips, stained with Sytox Green, and treated with the mouse monoclonal antihistone H2A-H2B-DNA complex antibody (gift of Dr. Volker Brinkmann, Max Planck Institute for Infection Biology, Berlin) according to Fuchs et al. (8 (link)). The preparations were observed using a Leica DM5000B microscope and Olympus FluoView FV1000 confocal microscope. To further confirm that DNA is the main structural component of METs, macrophages were incubated with C. albicans, as described previously, but in the presence of DNase (100 U/ml) and protease (trypsin, 0.25%) at the time of infection. After fixation, cells were stained with Sytox Green or with the Hemacolor staining protocol, as previously described.
4
Quantitative Fluorescence Microscopy Protocol
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Fluorescent signal was detected and visualised using a Leica DM5000 B microscope or a Leica SP8 confocal system. 3-D confocal stacks were acquired from tissues transduced with RGB vectors using the laser scanning function of the Leica SP8 confocal microscope with a 20x 0.75NA objective at a resolution of 1.3 pixels per mm. Z stacks were acquired at 1.14 mm per step in the Z plane from either 35 mm or 80 mm tissue sections. Confocal tile scanning of whole sagittal tissue sections was carried out using the Leica SP8 confocal microscope using the spiral scan function in Leica LAS-X software program. Brightfield images of DAB and AP staining were obtained using a Leica DM5000 B microscope or an Olympus VS110 slide scanner at a resolution of 2.9 pixels per mm.
Images were analysed using Fiji analysis software. 56 (link) The CellCounter plugin was used to record the number of cells and their spatial coordinates (XY) on at least 3 sections per animal. For each antigenic marker, the cell density (cells/mm 2 ) was calculated as the overall cell number divided by the area of the ROI (mm 2 ) (n=3 sections/mouse). For analysis of double positive cells, the deconvolution tool was used for separation of the chromogenic markers, DAB and AP. Delineated boundaries were saved as an ROI file and the spatial coordinates of each boundary were exported using the ROI manager tool in Fiji.
Images were analysed using Fiji analysis software. 56 (link) The CellCounter plugin was used to record the number of cells and their spatial coordinates (XY) on at least 3 sections per animal. For each antigenic marker, the cell density (cells/mm 2 ) was calculated as the overall cell number divided by the area of the ROI (mm 2 ) (n=3 sections/mouse). For analysis of double positive cells, the deconvolution tool was used for separation of the chromogenic markers, DAB and AP. Delineated boundaries were saved as an ROI file and the spatial coordinates of each boundary were exported using the ROI manager tool in Fiji.
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