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Leica dmi 6000b fluorescence microscope

Manufactured by Leica camera
Sourced in Germany

The Leica DMI 6000B is a fluorescence microscope designed for advanced imaging applications. It features a fully motorized system and supports a range of fluorescence techniques, including widefield, confocal, and high-speed imaging. The microscope is equipped with a high-sensitivity camera and a selection of filters to enable detailed analysis of fluorescently labeled samples.

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6 protocols using leica dmi 6000b fluorescence microscope

1

Quantifying Cell Surface Receptors

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A total of 3 × 105 cells were attached to poly-l-lysine (50 μg/mL)-coated coverslips and fixed with formaldehyde 4% (Sigma), blocked with γ-globulin (10 μg/mL) and stained with Hoechst33342 and HRH1 antibody (Antibodies-online GmbH, Aachen, Germany). Samples were observed in a Leica DMI6000 B fluorescence microscope (Leica, Wetzlar, Germany) and the images were analysed with Image J.
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2

Autophagy Quantification in Prostate Cancer Cells

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The detection of autophagic vacuoles was performed following the method described by Machado et al. [30 (link)], as well as the specifications provided by the manufacturer of the Autophagy Assay Kit (ab139484, Abcam, Cambridge, UK). PC-3 and DU145 cells (1 × 104) were seeded in a 96-well plate, stabilized for 24 h, and treated for further 24 h with sulforaphane and/or vitamin D. Fluorescence was recorded in a Synergy 2 spectrophotometer (BioTek®; Winooski, VT, USA) using Excitation/Emission filters of 463/534 nm and 350/461 nm. The results are presented as the autophagy ratio proposed by Tusskorn et al. [31 (link)]. The images were acquired with the Leica DMI 6000B Fluorescence Microscope (Leica; Wetzlar, HE, Germany) from Confocal Microscopy Multiuser Laboratory (LMMC).
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3

Autophagic Vesicle Quantification Assay

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The assay was performed as specified by the Autophagy Assay Kit’s manufacturer (ab139484; Abcam, Cambridge, UK). The HepG2 cells (1 × 104 cells/well) were seeded in a 96-well plate, stabilized for 24 h, and treated for further 24 h. Fluorescence was recorded in a Synergy 2 spectrophotometer (BioTek®; Winooski, VT, USA) with excitation/emission filters of 463/534 nm (green) and 350/461 nm (blue). The green fluorophore labels the autophagic vesicles that contain the LC3 protein while the blue fluorophore labels the cell nucleus. Fluorescence values are expressed as the autophagy ratio (ratio of green to blue fluorescence values) (Tusskorn et al., 2019). Images were acquired using the Leica DMI 6000B Fluorescence Microscope (Leica; Wetzlar, Hessen, Germany).
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4

Colocalization of NTCP Constructs in HEK293 Cells

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For colocalization studies, GFP- and mScarlet-tagged NTCP constructs were transiently transfected into HEK293 cells as described (Müller et al., 2018 (link)). Cells were seeded into µ-Slide chambered coverslips (Ibidi, Martiensried, Germany) and transfected with 0.25 µg of each appropriate construct pDNA using Lipofectamine 2000 (Thermo Fisher Scientific). Finally, 50 µl of the Lipofectamine-pDNA-complex were added to the cells before incubation was started at 37°C for 2 days. Staining of the nuclei was performed with Hoechst33342 (1 µg/ml, Thermo Fisher Scientific). Fluorescence microscopy was performed on a Leica DMI6000 B fluorescence microscope (Leica, Wetzlar, Germany). All images were taken and analyzed with the Leica software LAS X.
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5

Evaluation of ROS Production in Co-Cultures

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Dihydroethidium (DHE, Sigma Aldrich), an oxidative fluorescent dye, was used to evaluate in situ ROS production in a six-well cell culture plate containing the co-culture as previously described [21 (link)], with some modifications. Briefly, the unfixed co-culture was washed with phosphate buffer for 5 min, incubated with 500 µL DHE (10 µmol/L) for 1 h at room temperature in a dark chamber, and washed again three times with phosphate buffer. Each co-culture was then examined by fluorescence microscopy (Leica DMI 6000B Fluorescence Microscope) and the images were captured at 400× magnification. Red fluorescence superoxide (O2.-) and non-superoxide production were evaluated using the Image J software.
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6

Live/Dead Cell Viability Assay

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Cell viability was assessed using the LIVE/DEAD™ assay kit (Thermo Scientific; Canoga Park, CA, USA). HepG2 and HUVEC cells were seeded (5 × 103 cells/well) in 96-well black plates, stabilized for 24 h, and treated for 72 h. After treatment, the cultures were washed with PBS and stained according to the manufacturer’s recommendations. Calcein AM retained in living cells produces an intense green fluorescence, while ethidium homodimer (EthD-1) enters cells with damaged membranes and produces a red fluorescence in dead cells. Fluorescence was recorded in a Synergy H1 plate reader (BioTek®; Winooski, VT, USA) with excitation/emission filters of 485/530 and 530/645 nm for calcein and EthD-1, respectively. The fluorescence values of the negative control were considered as 100% cell viability, and the results were expressed as percentage (%) of viable cells. Images were captured using the Leica DMI 6000B Fluorescence Microscope (Leica; Wetzlar, Hessen, Germany).
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