CM-H2DCFDA (DCF-DA), which functions as a ROSsensitive fluorophore, was used to detect intracellular ROS generation. Mouse hepatocytes on 35-mm cell culture dishes (BD Bioscience) were treated with H2O2 with or without pretreatment with BHA or NAC for 30 min and cultured at 37℃ for 2 h. The cells were moved into the dark and incubated with 5 µM DCF-DA for 30 min at 37℃. Next, the cells were washed three times with PBS and viewed using fluorescence microscopy (DM IL LED Fluo; Leica, Germany) at 100 × magnification. Fluorescence intensity was measured using Tecan Infinite M200 Pro (Life Technologies).
Dm il led fluo
Manufactured by Leica
Sourced in Germany
The DM IL LED Fluo is a high-performance inverted microscope designed for fluorescence imaging applications. It features an LED illumination system, providing stable and long-lasting illumination for a variety of fluorescent dyes and probes.
Automatically generated - may contain errors
Lab products found in correlation
Las x software program, by Leica (3 mentions)
Live dead viability cytotoxicity kit, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Live dead kit, by Thermo Fisher Scientific (2 mentions)
35 mm cell culture dishes, by BD (1 mentions)
Superk extreme exw 12, by NKT Photonics (1 mentions)
Application suite x software, by Leica (1 mentions)
Alexa fluor 594 donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Leo 1450vp microscope, by Zeiss (1 mentions)
Eclipse te2000 e, by Nikon (1 mentions)
Mak044, by Merck Group (1 mentions)
Oil red o, by Merck Group (1 mentions)
Ad nc, by Genechem (1 mentions)
Lab tek chamber slide, by Thermo Fisher Scientific (1 mentions)
Dfc450c, by Leica (1 mentions)
Cellulase from trichoderma reesei, by Merck Group (1 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Ethidium homodimer 1, by Abcam (1 mentions)
Calcein am, by Merck Group (1 mentions)
46 protocols using dm il led fluo
1
Measurement of Intracellular ROS in Hepatocytes
2
STED Microscopy Imaging Protocols
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Confocal and STED images were acquired using a Leica SP8 STED 3X equipped with a SuperK Extreme EXW-12 (NKT Photonics) pulsed white light laser as an excitation source and a Onefive Katana-08HP pulsed laser as depletion light source (775-nm wavelength). All images were acquired using either a HC PL APO 63×/1.2 water objective, HC PL APO 86×/1.2 water CS2 objective, HC PL APO 63×/1.40-0.60 oil objective, or a HC PL APO 100×/1.40 NA oil immersion CS2 objective. Application Suite X software (LAS X; Leica Microsystems) was used to control imaging parameters. ATTO532 was imaged with 532-nm excitation. ATTO594 was imaged with 585-nm excitation and 775-nm depletion wavelengths. ATTO647N was imaged with 647-nm excitation and 775-nm depletion wavelengths. DY634 was imaged with 634-nm excitation. SYTOX Green and MitoTracker Orange were excited by 488-nm and 555-nm excitation light, respectively.
Widefield images to measure protein retention were obtained with a Leica tissue culture microscope (DM IL LED FLUO) equipped with a 10×/0.22 NA air objective. An Andor Clara CCD camera operated by MicroManager was used to record images.
Widefield images to measure protein retention were obtained with a Leica tissue culture microscope (DM IL LED FLUO) equipped with a 10×/0.22 NA air objective. An Andor Clara CCD camera operated by MicroManager was used to record images.
3
Microscopy Quantification of Stained Cells
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Microscope images were obtained using the Leica DM IL LED Fluo and Leica LAS X Software Program. Then, 4–8 images per well were taken and quantified for each condition and timepoint using ImageJ’s plugin Multipoint and Cell Counter feature to count the positively-stained cells by a double blinded approach.
4
Immunofluorescence Analysis of pSmad Signaling
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After five days of cultivation, hMSCs cultured on the MP-WH and MP-WH/BMP2 granules were rinsed with PBS and treated with 4% paraformaldehyde. Anti-pSmad 1/5/8 rabbit IgG (Abcam, Cambridge, UK) and Alexa Fluor 594 donkey anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA) were used to determine the pSmad expression. The nuclei were stained with a 4′,6-diamidino-2-phenylindole (DAPI) dye solution. The samples were observed using a fluorescence microscope (DM IL LED Fluo, Leica, Microsystems, Wetzlar, Germany).
5
Microscopic Characterization of Polymeric Microbeads
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Before freeze-drying, a sample of each PMBs batch was transferred to a glass slide for direct observation with a fluorescence microscope (DM IL LED Fluo - Leica, Germany). PMBs were also examined under a confocal microscope (Eclipse TE2000-E - Nikon, 94504 Champigny sur Marne, France) using a 488-nm laser and Z-stack images with a 3-μm step were obtained. Scanning Electronic Microscopy (SEM) was carried out using a LEO 1450VP microscope (Zeiss) by detecting secondary electrons at the following settings: 5 keV–13 pA. Samples were previously allowed to slowly dry at room temperature for a week and gold/palladium coated using a Desk III metal coater (Denton).
6
Phagocytosis Assay with Rhinosectan
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The Cayman Phagocytosis Assay Kit (IgG FITC) (Cayman Chemical Company, USA) employs latex beads coated with fluorescently-labelled rabbit-IgG as a probe for the identification of factors regulating the phagocytic process in vitro.
The assay was performed following the manufacturer’s instructions. Briefly, 100 μl of Latex Beads-Rabbit IgG-FITC solution (diluted 1:10 in saline solution) was added simultaneously with 30 μl of Rhinosectan® or saline solution to MucilAir™ cell monolayers. After incubation for 15 or 60 min at 37 °C, the supernatant was carefully aspirated and cells were gently washed with assay buffer to remove unbound beads. For the qualitative analysis, cells were analyzed directly with the inverted fluorescence microscope (Leica DM IL LED FLUO, Germany) with a10× objective, which was equipped with a filter capable of measuring excitation and emission at 485 and 535 nm, respectively. For the quantitative analysis, the fluorescence intensity of the cells was measured in a fluorescence plate reader with the appropriate filters (ex/em 485/535 nm) (Spectrophotometer Infinite M200, Tecan, Switzerland). Two biological replicates for each treatment (negative control and Rhinosectan®) were used for fluorescence evaluation.
The assay was performed following the manufacturer’s instructions. Briefly, 100 μl of Latex Beads-Rabbit IgG-FITC solution (diluted 1:10 in saline solution) was added simultaneously with 30 μl of Rhinosectan® or saline solution to MucilAir™ cell monolayers. After incubation for 15 or 60 min at 37 °C, the supernatant was carefully aspirated and cells were gently washed with assay buffer to remove unbound beads. For the qualitative analysis, cells were analyzed directly with the inverted fluorescence microscope (Leica DM IL LED FLUO, Germany) with a10× objective, which was equipped with a filter capable of measuring excitation and emission at 485 and 535 nm, respectively. For the quantitative analysis, the fluorescence intensity of the cells was measured in a fluorescence plate reader with the appropriate filters (ex/em 485/535 nm) (Spectrophotometer Infinite M200, Tecan, Switzerland). Two biological replicates for each treatment (negative control and Rhinosectan®) were used for fluorescence evaluation.
7
Quantification of AhR Ligand in Hepatocytes
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Hepatocytes were fixed with an ethanol:PBS (60:40, v/v) solution for 10 min at room temperature. Cells were permeabilized with 0.05% Triton X-100 for 10 min and treated with the anti-AhR (1:50) (BD Biosciences, Franklin Lakes, NJ, USA) antibody overnight at 4 °C. Cells were imaged with an inverted fluorescence microscope (Leica DM IL LED Fluo, Wetzlar, Germany). Arbitrary units of the AhR ligand in hepatocytes were determined using ImageJ software (version 1.43 n).
8
Quantifying Intracellular Reactive Oxygen
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Cells were incubated with fluorescent ROS probe dichloro-dihydro-fluorescein diacetate (DCFH-DA, S0033M-1, Beyotime, Shanghai, China) for 30 min. Then, cells were washed with DPBS (14190144, Thermo Fisher Scientific, Waltham, MA, USA). A fluorescence microscope (DMIL LED fluo, Leica, Wetzlar, Germany) was applied to observe and capture cell images. ImageJ software was then used to calculate the integrated density and cell area, with the integrated density subsequently normalized by the cell area.
9
Multiparametric Microscopy Imaging Protocol
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Microscope two-dimensional images were obtained using the Leica DM IL LED Fluo and Leica LAS X Software Program. 2–3 two-dimensional images were captured per well at 48hpi for each condition. These images were quantified using Image J’s plugin (Multipoint and Cell Counter). The positively stained cells were counted by a double blinded approach. Confocal slide samples were imaged using a Leica SP8 MP-DIVE-FLIM Microscope at the Advanced Light Microscopy/Spectroscopy Laboratory and Leica Microsystems Center of Excellence at the California NanoSystems Institute at UCLA (RRID:SCR_022789) with funding support from NIH Shared Instrumentation Grant S10OD025017 and NSF Major Research Instrumentation grant CHE-0722519. Confocal three-dimensional images were collected in 1024×1024 format using a 63x oil immersion objective lens, fixed scan rate of 8000Hz, and averaged 12 times. Excitation laser lines and emission detection wavelengths were optimized for the fluorescent tags as follows: blue channel excitation of 405nm with emission detection range of 420–470nm, green channel excitation of 488nm with emission detection range of 500nm-530nm, and red channel excitation of 552nm with emission detection range 590–650nm.
10
Agarose-Based Pancreatic Tumoroid Formation
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The agarose gel-based matrices utilized in this study to create MIA PaCa-2
pancreatic tumoroids. 2.5 mL of 0.5%, 0.8%, and 1.5% agarose solutions were
prepared and heated in a microwave, and they were put into 33 mm diameter
Petri dishes for cell culture and allowed to polymerize. After that, each
Petri dish received 2.5 mL of the culture medium (DMEM + 10% FBS) and was
maintained for 1 day at 37 °C.
A total of 100 cells in 10 μL volumes were pipetted into the dishes, it was
allowed to absorb the solution by the agarose matrices for 2 h, then a cell
culture medium was added to the cells and the Petri dishes were placed in
the incubator with their bottoms facing up. Standard incubation conditions
were used for the cultures (5% CO2, 37 °C). The culture media
were replaced 4 days after the initial seeding, and this process was
repeated every 2 days after that. The plates were removed from the incubator
every 2 days to observe the development of tumoroids with an inverted phase
contrast microscope (Leica, Dmil Led Fluo). The imaging of 3 tumoroid
cultures with 0.5%, 0.8%, and 1.5% concentrations was initiated with the
mini-Opto tomography imaging system (Figure 2b , c) after observing the
effective formation and proper growth of the tumoroid by the inverted phase
contrast microscope (Figure 2a ).
pancreatic tumoroids. 2.5 mL of 0.5%, 0.8%, and 1.5% agarose solutions were
prepared and heated in a microwave, and they were put into 33 mm diameter
Petri dishes for cell culture and allowed to polymerize. After that, each
Petri dish received 2.5 mL of the culture medium (DMEM + 10% FBS) and was
maintained for 1 day at 37 °C.
A total of 100 cells in 10 μL volumes were pipetted into the dishes, it was
allowed to absorb the solution by the agarose matrices for 2 h, then a cell
culture medium was added to the cells and the Petri dishes were placed in
the incubator with their bottoms facing up. Standard incubation conditions
were used for the cultures (5% CO2, 37 °C). The culture media
were replaced 4 days after the initial seeding, and this process was
repeated every 2 days after that. The plates were removed from the incubator
every 2 days to observe the development of tumoroids with an inverted phase
contrast microscope (Leica, Dmil Led Fluo). The imaging of 3 tumoroid
cultures with 0.5%, 0.8%, and 1.5% concentrations was initiated with the
mini-Opto tomography imaging system (
effective formation and proper growth of the tumoroid by the inverted phase
contrast microscope (
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