Leica dmi6000 b inverted microscope

Manufactured by Leica Microsystems

Sourced in Germany, Canada, United Kingdom

The Leica DMI6000 B is an inverted microscope designed for cell and tissue culture applications. It features a stable, vibration-free stand and a broad range of optical accessories to support various imaging techniques, including brightfield, phase contrast, and fluorescence microscopy.

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39 protocols using leica dmi6000 b inverted microscope

Visualizing Autophagosome Dynamics in Microscopy

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Animals were mounted in 3 μl of 15 mM tetramisole (Sigma-Aldrich, L9756) in M9 buffer suspension containing 45-μm polystyrene microspheres (Polysciences, 07314-5), and sealed between 2 coverslips (Corning, 2845-18 and 2975-246) with vaseline. Alternatively, for time-lapse recordings (Movies S1, S2 and S3), a recently described procedure was used.⁷⁵ (link) Microscopy was performed with a VisiScope spinning disk confocal microscope system (Visitron Systems, Puchheim, Germany) based on a Leica DMI6000B inverted microscope (Leica Microsystems, Wetzlar, Germany), a Yokogawa CSU X1 scan head (Yokogawa Electric Corporation, Tokyo, Japan), and a Hamamatsu ImagEM EM-CCD (Hamamatsu Photonics, Hamamatsu, Japan). All acquisitions were performed at 21°C to 23°C using a Leica HC PL APO 40x/1.3 oil or a Leica HC PL APO 63x/1.4–0.6 oil objective.
Animals stained with DAPI were imaged using a Leica DM IL epifluorescence microscope (Leica Microsystems, Wetzlar, Germany) equipped with a Leica HC PL FL 10×/0.3 objective and a Motic Moticam 1SP camera (Motic Deutschland GmbH, Wetzlar, Germany) with Motic acquisition software.
3D reconstructions were performed using imod (http://bio3d.colorado.edu/imod/) for cell outlines and Endrov (http://www.endrov.net/) for autophagosome movement.

Gomes L.C., Odedra D., Dikic I, & Pohl C. (2016). Autophagy and modular restructuring of metabolism control germline tumor differentiation and proliferation in C. elegans. Autophagy, 12(3), 529-546.

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3D Deformation Tracking of Cell Substratum

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The 3-D deformation of the substratum at two different planes beneath the surface, z = h₀ and z = h₁, was measured by tracking respectively the red and yellow fluorescent beads. We acquired time-lapse sequences of image z-stacks using a Leica DMI6000 B inverted microscope (Leica Microsystems, Inc., Buffalo Grove, IL) equipped with a Zyla 4.2 sCMOS camera (Andor Technology Ltd., Belfast, UK) and a 20x objective lens. The imaging setup was controlled by the open source microscopy software Micro-Manager40 41 . Each z-stack consisted of 20 images at 0.5 μm increments, and was vertically centered at the plane of maximum fluorescence intensity as determined by the autofocus system. The position and shape of the cell at each instant of time was recorded with an additional bright-field phase contrast image. We acquired the two fluorescent z-stacks and the phase-contrast image of the cell every 60 seconds.

Álvarez-González B., Zhang S., Gómez-González M., Meili R., Firtel R.A., Lasheras J.C, & del Álamo J.C. (2017). Two-Layer Elastographic 3-D Traction Force Microscopy. Scientific Reports, 7, 39315.

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Immunohistochemical Analysis of NXF3 Expression

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Tissue sections (4 μm) were stained with hematoxylin and eosin for histological analysis and with specific primary anti-human antibodies against NXF3 (1:150; LS-C31687; LifeSpan Biosciences, Inc., Seattle, WA, USA) for IHC. Following microwave antigen retrieval, the tissues were incubated with the primary antibodies overnight at 4°C followed by a 30-min incubation with the secondary antibody (Dako EnVision kit, Dako, Glostrup, Denmark). The reaction was visualized with diaminobenzidine and the tissues were counterstained with hematoxylin.
The tissue sections were viewed at ×200 magnification using a Leica DMI6000B inverted microscope (Leica Microsystems, Heidelberg, Germany) and images were captured. Two experienced pathologists independently assessed all IHC staining. The scoring for nuclear NXF3 expression was based on the staining proportion and intensity. The staining proportion was scored as follows: 0–25% staining, 1; 26–50% staining, 2; 51–75% staining, 3; and 76–100% staining, 4. The staining intensity was scored as follows: Negative intensity, 0; weakly positive, 1; moderately positive, 2; and strongly positive, 3, according to a previous study (12 (link)). The sum of the proportion and intensity scores was used to calculate the final staining score, which was then categorized as low (1 (link)–5 (link)) or high (6 (link)–7 (link)).

JIANG J.H., GAO Q., KE A.W., YU Y., SHI G.M., FAN J., ZHOU J, & HUANG X.W. (2014). Prognostic significance of nuclear RNA export factor 3 in hepatocellular carcinoma. Oncology Letters, 7(3), 641-646.

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Quantifying Autophagic Flux in Live Cells

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Cyto-ID selectively labels accumulated autophagic vacuoles monitoring autophagic flux in live cells. Cells were seeded on 6-well plates 24 h prior to treatment. After 24 or 48-h treatments, cells were washed with 1X assay buffer provided in the Cyto-ID autophagy detection kit (Enzo Life Sciences Inc., Cat. No. 51031, Farmingdale, NY, USA), and incubated in half 1:1000 diluted Cyto-ID green detection reagent in Hank’s Balanced Salt Solution and half cell culture media for 30 min at 37 °C. Next, (a) for image-based cytometry, cells were washed and resuspended in 1X assay buffer. A TALI Image-Based Cytometer (Life Technologies Inc., Cat. No. T10796, Burlington, ON, Canada) was utilized to quantify the percentage of cells fluorescing green (containing autophagic vacuoles). Cells from 18 random fields were used to analyze the green (ex. 458 nm; em. 525/20 nm) channel. OR (b) for fluorescent imaging, cells were washed and resuspended in 1X PBS, incubated with 10 µM Hoechst 33342 (Molecular Probes, Cat. No. H3570, Eugene, OR, USA) for 10 min at 37 °C, and imaged using epifluorescence microscopy on a Leica DMI6000 B inverted microscope (Leica Microsystems, Concord, ON, Canada). Fluorescence quantification was performed on the images using ImageJ software version 1.52.

Wear D., Bhagirath E., Balachandar A., Vegh C, & Pandey S. (2023). Autophagy Inhibition via Hydroxychloroquine or 3-Methyladenine Enhances Chemotherapy-Induced Apoptosis in Neuro-Blastoma and Glioblastoma. International Journal of Molecular Sciences, 24(15), 12052.

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Immunofluorescence Analysis of Autophagy and Oxidative Stress

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Cells were seeded on 8-chamber slides (Bio Basic Canada Inc., Cat. No. SP41219, Markham, ON, Canada) 24 h prior to treatment. Following treatments of 24 or 48 h, cells were fixed with 3.7% formaldehyde prepared in 1X PBS for 15 min at room temperature, permeabilized with 0.15% Triton X-100 for 2 min and blocked with 5% bovine serum albumin for 1 h. Cells were washed with 1X Tris-Buffered Saline, 0.1% Tween 20 Detergent (TBST) and incubated for 1 h at room temperature with the following primary antibodies: LAMP1 (mouse IgG, 1:250, Cat. No. ab25630) (Abcam Inc., Cambridge, UK), LC3B (rabbit IgG, 1:500, Cat. No. ab192890) (Abcam Inc.), 4-HNE (rabbit IgG, 1:200, Cat. No. ab46545) (Abcam Inc.). Cells were washed with TBST and incubated with goat anti-rabbit Alexa Fluor™ 568 (1:500, Thermo Scientific Canada, Cat. No. A11011) and/or horse anti-mouse fluorescein isothiocyanate (1:500, MJS BioLynx Inc., Brockville, Canada, Cat. No. Fl-2000) secondary antibodies for 1 h at room temperature. Cells were washed with TBST and incubated for 2 min with 10 µM Hoechst 3342 (Molecular Probes, Cat. No. H3570, Eugene, OR, USA). Cells were washed with 1X PBS and imaged via epifluorescence microscopy with a Leica DMI6000 B inverted microscope (Leica Microsystems, Concord, ON, Canada). ImageJ software was utilized for fluorescent quantification.

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Senescence-associated β-Galactosidase Assay

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An SA β-galactosidase staining kit was purchased from Beyoncé Biotechnology Co (Shanghai, China). The SA β-galactosidase staining kit was used to detect the activity of senescence-specific β-galactosidase. The cells were inoculated in 96-well plates for 24 h. The cell culture was aspirated, washed once with PBS, treated with 100 μL of β-galactosidase staining fixative and fixed for 15 min at room temperature. The 1000 μL cell-staining working solution consisted of 10 μL β-galactosidase staining solution A, 10 μL β-galactosidase staining solution B, 930 μL β-galactosidase staining solution C and 50 μL X-Gal solution. After the cell fixative was aspirated, the cells were washed with PBS thrice for 3 min each time. Then, we aspirated the PBS and added 100 μL of staining working solution to each well. We sealed the 96-well plates using a sealing film and incubated them overnight at 37 °C in a CO₂-free incubator for observation under a Leica DMI6000 B inverted microscope (Leica Microsystems, Weztlar, Germany). Cell counts were performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA), and quantitative results were obtained by dividing the number of positive cells in the field of view by the total number of cells.

Hao W., Shan W., Wan F., Luo J., Niu Y., Zhou J., Zhang Y., Xu N, & Xie W. (2023). Canagliflozin Delays Aging of HUVECs Induced by Palmitic Acid via the ROS/p38/JNK Pathway. Antioxidants, 12(4), 838.

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Histological analysis of mouse submandibular gland

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mSMG tissues were immersed in 10% formalin at room temperature for one day, dehydrated in serial ethanol solutions (50%, 70% and 100% for 2 h each), embedded in paraffin wax and cut into 7 μm sections. mSMG sections from each group were deparaffinized with xylene and rehydrated with serial ethanol solutions (100%, 70% and 50%) and distilled water. Then, hematoxylin and eosin (H&E) and picrosirius red staining were performed, and tissue sections were subjected to a blind histopathological analysis using a Leica DMI6000B inverted microscope (Leica Microsystems, Wetzlar, Germany) as well as an Olympus BX53 Light Microscope (Olympus America, Center Valley, PA). In addition, the ratio of acinar structures to ductal structures was analyzed using ImageJ and GraphPad Prism 6.

Nam K., Maruyama C.L., Wang C.S., Trump B.G., Lei P., Andreadis S.T, & Baker O.J. (2017). Laminin-111-derived peptide conjugated fibrin hydrogel restores salivary gland function. PLoS ONE, 12(11), e0187069.

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Tracking Co-cultured Human Fibroblasts and Endothelial Cells

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For live cell tracking, I-hVFFs were incubated with 10 μM Cell Tracker^™ Orange CMRA (Molecular Probes) for 45mins and washed 3 times with 1X D-PBS. Cells were then trypsinized and seeded at a density of 3 × 10⁴ cells/cm². After 48 hours, ECs were labeled with 10 μM Cell Tracker ^™ Green CMFDA (Molecular Probes) for 45 minutes, trypsinized and seeded directly on top of the I-hVFFs. Cultures were visualized using a Leica DMI6000 B inverted microscope with an EL6000 external light source (Leica Microsystems, Wetzler, Germany) 24 hours after seeding ECs using a ×10objective. A CooolSNAP HQ² camera and Leica application suite (LAS) AF6000 software (Leica Microsystems) were utilized for image acquisition. All images were taken using identical settings for exposure time. Images were edited using ImageJ (National Institutes of Health, Bethesda, MD).

Walimbe T., Panitch A, & Sivasankar M.P. (2016). An in-vitro scaffold-free epithelial-fibroblast coculture model for the larynx. The Laryngoscope, 127(6), E185-E192.

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Paraffin Embedding and H&E Staining

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Cell sheets were fixed in 4% PFA for 10 min, dehydrated in 70% ethanol solution, embedded in paraffin wax, and cut into 3 μm sections. Then, they were deparaffinized with xylene and rehydrated with serial ethanol solutions and distilled water. Finally, hematoxylin and eosin staining was performed, and specimens were examined using a Leica DMI6000B inverted microscope (Leica Microsystems, Wetzlar, Germany).

Nam K., Kim K., Dean S.M., Brown C.T., Davis R.S., Okano T, & Baker O.J. (2019). Using cell sheets to regenerate mouse submandibular glands. NPJ Regenerative Medicine, 4, 16.

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Multimodal Cellular Imaging Technique

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Cells were cultured on coverslips and treated as aforementioned. Cells were fixed in 4% paraformaldehyde for 10 min at room temperature prior to permeabilization in PBS containing 0.1% Triton X-100 for 2 min. MitoTracker Red CMXRos (Thermo Fisher Scientific, Inc.) was used for mitochondrial staining at 37°C for 30 min. Phalloidin-fluorescein isothiocyanate (Sigma-Aldrich; Merck KGaA) was used for F-actin staining at 37°C for 30 min. Cells were rinsed 3 times with PBS and incubated with DAPI (1 µg/ml; cat. no. 9542; Sigma-Aldrich; Merck KGaA) at 37°C for 30 min. Slides were analyzed by Leica DMi6000 B inverted microscope (Leica Microsystems, Inc.) according to manufacturer's instruction and 3 fields per view were analyzed.

Yang L., Hong Q., Xu S.G., Kuang X.Y., Di G.H., Liu G.Y., Wu J., Shao Z.M, & Yu S.J. (2019). Downregulation of transgelin 2 promotes breast cancer metastasis by activating the reactive oxygen species/nuclear factor-κB signaling pathway. Molecular Medicine Reports, 20(5), 4045-4058.

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