Micrographs of neuron-like SH-SY5Y cells were obtained using a DM IL LED fluorescence microscope (Leica Microsystems, Mannheim, Germany) with an appropriate excitation filter for the assay, or a brightfield setup with a DP73 high-performance digital camera (Olympus, Tokyo, Japan). Since the signal-to-noise ratio for staining was moderate, the contrast was slightly adjusted in ImageJ software (Fiji) without affecting the resulting observation. Original images are included in the Supplementary Materials .
Dm il led fluorescence microscope
Manufactured by Leica
Sourced in Germany
The DM IL LED fluorescence microscope is a compact and versatile microscope designed for routine fluorescence imaging. It features an LED illumination system for stable and efficient fluorescence excitation. The microscope is equipped with standard fluorescence filter cubes for common fluorophores and can be used for a variety of applications in life science research and clinical settings.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Cell light edu apollo 567, by RiboBio (1 mentions)
Bovine serum albumin (bsa), by Avantor (1 mentions)
Fluoroskan fl, by Thermo Fisher Scientific (1 mentions)
Pkh67 green fluorescent cell linker midi kit, by Merck Group (1 mentions)
Ethidium homodimer 1, by Thermo Fisher Scientific (1 mentions)
Phalloidin ifluor 555, by Abcam (1 mentions)
Goat anti mouse alexa fluor 488, by Abcam (1 mentions)
Flexstation 3, by Molecular Devices (1 mentions)
Nova nanosem 230, by Thermo Fisher Scientific (1 mentions)
Thunder 3d live cell, by Leica (1 mentions)
Phalloidin tritc, by Merck Group (1 mentions)
Cellrox green reagent, by Thermo Fisher Scientific (1 mentions)
Application suite, by Leica (1 mentions)
15 protocols using dm il led fluorescence microscope
1
Imaging Neuron-like SH-SY5Y Cells
2
EdU Incorporation Analysis in Colorectal Cancer Cells
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EdU incorporation was performed using the Cell Light™ Edu Apollo 567 in vitro kit (Guangzhou RiboBio Co., Ltd.) according to the manufacturer's protocol. A total of 5x103 SW480 and SW620 cells/well were plated into 96-well plates in RPMI-1640 medium, containing 10% FBS. Following overnight attachment in a humidified incubator at 37˚C with 5% CO2, SW480 and SW620 cells were transfected with siRNA-KRT80 or siRNA-NC for 48 h. Subsequently, the cells were incubated with 100 µl 50 µM EdU reagents at 37˚C. Following 2 h of incubation, the cells were fixed with 50 µl 4% paraformaldehyde for 30 min at 25˚C. Cells were washed three times in 100 µl PBS -0.1% Triton X-100 prior to incubation with 100 µl 1x Apollo solution (containing deionized water 93.8 µl, Apollo® reaction buffer 5 µl, Apollo® catalyst solution 1 µl, Apollo® fluorescent dye solution 0.3 µl and Apollo® buffer additive 0.9 mg) for 30 min at room temperature in the dark. The cells were subsequently stained with 100 µl 1X Hoechst 33342 nuclear dye for 30 min at room temperature. Stained cells were visualized using a DM IL LED fluorescence microscope (magnification, x200; Leica Microsystems GmbH), with an excitation wavelength of 567 nm. Image analysis was performed using ImageJ v1.8.0 software (National Institutes of Health).
3
Immunofluorescence Staining of PEDV-Infected Cells
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Vero cells grown on microscope coverslips placed in 6-well tissue culture plates were infected with parental (P5) and rescued (P10) PEDVs at a multiplicity of infection (MOI) of 0.1 for 1 h. At 24 h post-infection (hpi), infected cells were fixed with 4% paraformaldehyde for 10 min at room temperature (RT) and permeabilized with 0.2% Triton X-100 in PBS at RT for 10 min. The cells were blocked with 1% bovine serum albumin in PBS at RT for 30 min and then stained with PEDV N- or ORF3-specific MAb for 2 h. After washing five times in PBS, the cells were incubated at RT for 1 h with the Alexa Fluor 488-conjugated or 594-conjugated (only for icPEDV-EGFP) secondary antibody, followed by counterstaining with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). The coverslips were mounted on glass microscope slides in a mounting buffer, and the stained cells were visualized under a Leica DM IL LED fluorescence microscope (Leica, Wetzlar, Germany).
4
Immunofluorescence Staining of Cultured Cells
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PK-rS1-cFc cells were grown on microscope coverslips placed in 6-well tissue culture plates. At 48 h post-seeding, the cells were fixed with 4 % paraformaldehyde for 10 min at room temperature (RT) and permeabilized with 0.2 % Triton X-100 in phosphate-buffered saline (PBS) at RT for 10 min. The cells were subsequently blocked with 1 % bovine serum albumin (Amresco, Solon, OH, USA) in PBS for 30 min at RT and then incubated with a goat anti-chicken IgG antibody conjugated to FITC. Finally, the cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma, St. Louis, MO, USA), and cell staining was visualized using a Leica DM IL LED fluorescence microscope (Leica, Wetzlar, Germany).
5
SARS-CoV-2 Immunofluorescence Assay
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Vero E6 cells grown in 96-well tissue culture plates were mock infected or infected with SARS-CoV-2 (P3) at a multiplicity of infection (MOI) of 1 for 1 h and cultured under the indicated conditions. At 24 h postinfection (hpi), SARS-CoV-2-infected cells were fixed with 4% paraformaldehyde for 10 min at room temperature and permeabilized with 0.2% Triton X-100 in PBS at room temperature for 10 min. The cells were blocked with 1% bovine serum albumin in PBS at room temperature for 30 min and then stained with the SARS-CoV-2 N-specific MAb. After washing five times in PBS, the cells were incubated at room temperature for 1 h with the corresponding Alexa Fluor–conjugated secondary antibodies, followed by counterstaining with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). The coverslips were mounted on glass microscope slides in a mounting buffer, and the stained cells were visualized under a Leica DM IL LED fluorescence microscope (Leica, Wetzlar, Germany).
6
Quantifying PEDV Infection in Vero Cells
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Vero cells at 2 × 104 cells/well were grown in 96-well tissue culture plates for 24 h and were mock infected or infected with icPEDV-EGFP-P10 at an MOI of 0.1. At the indicated time points, the cells were examined with a Leica DM IL LED fluorescence microscope, and pictures of the EGFP fluorescence images were obtained. Subsequently, the fluorescence intensity of EGFP was measured at 485/538 nm using a microplate fluorometer (Fluoroskan FL; Thermo Scientific, Waltham, MA, USA).
7
Fluorescent Labeling and Uptake of Extracellular Vesicles
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EVs were labeled following the instructions of the PKH67 Green Fluorescent Cell Linker Midi Kit (MIDI67‐1KT, Sigma‐Aldrich, St. Louis, MO). Isolated EVs were mixed with 1 mL of diluent and 4 μL of PKH67 solution (both provided in the kit) and incubated for 4 min. Then, 2 mL of 0.5% bovine serum albumin in PBS was added for quenching. The labeled EVs were ultracentrifuged twice at 100 000 × g at 4°C for 70 min and resuspended in 100 μL of PBS. To examine EV uptake, LoVo or HT29 cells were seeded in a 24‐well plate at 1 × 104 cells/well and incubated at 37°C for 12 h in an atmosphere containing 5% CO2. Then, 10 μg of PKH67‐labeled EVs were added to each well and the plate was further incubated at 37°C in 5% CO2 for 2 or 12 h. The cells were fixed for 20 min with 4% paraformaldehyde and the nuclei were stained with Hoechst 33258, after which they were observed under a Leica DM IL LED fluorescence microscope using a 400× objective lens for EV uptake. The microscopy imaging parameters were set at initial acquisition and were kept constant between acquisitions. Green fluorescence represents PKH67 labeling of EVs.
8
Macrophage Survival after GBS Infection
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To measure the survival rate of macrophages 24 or 48 hours after the initial infection period, an ethidium homodimer-1 (Thermo Fisher Scientific) membrane permeabilization assay was used. After the macrophages were infected with GBS (MOI 10 for 1 hour) as before, they were washed and treated with 100µg/mL gentamicin and 5µg/mL penicillin G for 24-48 hours, as indicated for each experiment. They were then incubated with ethidium homodimer-1 (4 µM) in PBS, which is a fluorescent dye that enters dead cells by crossing their damaged membranes and then binding tightly to DNA. Macrophages were visualized by fluorescence microscopy (Leica DMIL LED fluorescence microscope with Ocular software (version 2); 20x or 40x objective, as indicated) to distinguish dead macrophages (bright red) from living macrophages (unstained). Images were captured from at least 6 biological replicates per condition, with at least 3 fields captured per well. At least 9,000 cells were counted per condition.
9
Visualization of Cytoskeleton in Hydrogel-Encapsulated MSCs
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After cultivation,
samples were fixed in 4% paraformaldehyde for 45 min and permeabilized
with 0.5% Triton-X in PBS for 30 min prior to staining with the phalloidin
staining solution. Samples were stained with 1 μg/mL Phalloidin
iFluor-555 (Abcam, Cambridge, United Kingdom) in PBS with 1% BSA,
to visualize the cytoskeleton arrangement of the hydrogel encapsulated
MSCs by staining of the actin filaments. Samples were then incubated
for 30 min at 37 °C in the dark, washed with PBS, and then inspected
using a DM IL LED fluorescence microscope (Leica, Wetzlar, Germany).
samples were fixed in 4% paraformaldehyde for 45 min and permeabilized
with 0.5% Triton-X in PBS for 30 min prior to staining with the phalloidin
staining solution. Samples were stained with 1 μg/mL Phalloidin
iFluor-555 (Abcam, Cambridge, United Kingdom) in PBS with 1% BSA,
to visualize the cytoskeleton arrangement of the hydrogel encapsulated
MSCs by staining of the actin filaments. Samples were then incubated
for 30 min at 37 °C in the dark, washed with PBS, and then inspected
using a DM IL LED fluorescence microscope (Leica, Wetzlar, Germany).
10
Virus Titer Determination by TCID50 Assay
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Titers of the virus stocks were determined by use of a 50% tissue culture infectious dose (TCID50) assay adapted for indirect detection of virus-infected cells. Briefly, tenfold viral dilutions were inoculated on Vero-E6 cells and incubated for 7 (DOBV, SEOV and TULV) or 12 days (PUUV) at 37°C, which corresponded to peak titers during virus culture (data not shown). Subsequently, cells were fixed using 4% PFA and permeabilized using 0.02% Triton X-100 in PBS. Subsequently, cells were washed with PBS and stained using mouse anti-hanta nucleoprotein (1:500; abcam, Cambridge, United Kingdom) (for PUUV, SEOV and TULV) or mouse anti-hantavirus Dobrava strain nucleoprotein (1:1,000; IBTsystems, Binzwangen, Germany) (for DOBV) as primary antibodies and goat anti-mouse-AlexaFluor488 (1:200; Abcam, Cambridge, United Kingdom) as a secondary antibody. Positive wells were visualized using a Leica DMIL LED fluorescence microscope and scored by eye. TCID50 titers were calculated using the Spearman and Kärber algorithm (Hierholzer and Killington, 1996 (link)).
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