For bioluminescence recordings, 35 mm FluoroDishes (World Precision Instruments) were prepared with a layer of autoclaved high-vacuum grease (Dow Corning) on the top of the rim and loaded with 1.2 ml recording medium [3.5 g/L DMEM, d -glucose (Sigma-Aldrich), 0.035% sodium bicarbonate, 10 mm HEPES, 1 mg/ml penicillin-streptomycin, 5% B27, and 0.1 mm luciferin in autoclaved Milli-Q water] and a Millicell insert (Millipore) under sterile conditions. Tissue explants were microdissected from slices and placed on the Millicell insert. The dish was sealed by a glass coverslip. Prepared dishes were placed in a 37°C chamber and imaged with a self-contained Olympus LV200 luminescence microscopy system fitted with a cooled Hamamatsu C9100-13 EM-CCD camera using a 20× 0.45 NA LUCPLFLN objective (Olympus). Bright-field (bf) pictures were taken before and after recording of the bioluminescence signal in darkness. This was to reposition the slice following drug treatment and to confirm the regions-of-interest (ROIs). The recording parameters (EM: 4, gain: 1, exposure time: 1800 s) were identical for Fbxl3+/+ and Fbxl3Afh/Afh cultures. Treatments were performed by transferring the insert containing the tissue explant to a new culture dish containing fresh media with either vehicle or drug.
Fluorodishes
Manufactured by World Precision Instruments
Sourced in United States
FluoroDishes are specialized cell culture dishes designed for fluorescence microscopy applications. They feature a thin, optically clear glass or plastic bottom that allows for high-quality imaging of fluorescently labeled cells or samples.
Automatically generated - may contain errors
Lab products found in correlation
Axiovert 200m, by Zeiss (6 mentions)
Rnaimax, by Thermo Fisher Scientific (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Live cell imaging solution, by Thermo Fisher Scientific (3 mentions)
Neurobasal, by Thermo Fisher Scientific (2 mentions)
Image pro plus software, by Media Cybernetics (2 mentions)
Spinning disk confocal microscope, by Nikon (2 mentions)
On targetplus smartpool sirna, by Horizon Discovery (2 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
High vacuum grease, by Dow (1 mentions)
C9100 13 em ccd camera, by Olympus (1 mentions)
Millicell insert, by Merck Group (1 mentions)
Lucplfln objective, by Olympus (1 mentions)
D glucose, by Merck Group (1 mentions)
P4707, by Merck Group (1 mentions)
Eclipse te2000 u inverted microscope, by Nikon (1 mentions)
Eclipse ez c1 system, by Nikon (1 mentions)
Fluoro carbonyl cyanide phenylhydrazone (fccp), by Merck Group (1 mentions)
Bcecf am, by Thermo Fisher Scientific (1 mentions)
Plan neofluor, by Zeiss (1 mentions)
35 protocols using fluorodishes
1
Real-time Bioluminescence Imaging Assay
2
Cell Culturing on Glass Dishes
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Cells were cultured in MEM complemented with 10% FBS, 1% Pen/Strep, 1% L-glutamine, and 1% nonessential amino acids. Cells were seeded for 24 h at low concentration on glass bottom FluoroDishes of 25 mm and 0.17 mm thickness (World Precision Instruments Inc., Sarasota, FL, USA). For mESCs, FluoroDishes were first coated with Vitronectin following the manufacturer’s protocol.
3
Culturing Hippocampal Neurons for Transfection
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Hippocampal neurons were obtained from E18 rat embryos, plated at 120 × 103 on 35mm, 10mm well FluoroDishes (World Precision Instruments) coated with poly-D-lysine and laminin as described above, and maintained in enriched Neurobasal (ThermoFisher Scientific) medium as described above. Neurons were transfected at DIV6-DIV8 using Lipofectamine2000 (Life Technologies) and imaged 1 day later.
4
VAMP7-pHluorin Exocytosis Dynamics
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Non-patterned cells were seeded in fluorodishes (World Precision Instrument) coated with fibronectin [or PLL (Merck P4707)]. DMEM/F12 media + 20 mM HEPES was used for imaging. Patterned cells were prepared as described. The acquisition was made using an inverted Nikon TIRFM equipped with an EMCCD camera (efficiency 95%) with a 100× objective (pixel size = 0.160 µm). The following lasers were used 491 nm, 561 nm, and 642 nm. Time-lapse of VAMP7-pHluorin was acquired with a frame rate of one image every 300 ms during 5 min. Frame rate was set according to the half-life of exocytosis events. Due to microscopic device delay, the actual frame rate was computed using the computer time of saved files.
5
Live Cell Imaging of Fluorescent Proteins
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For video microscopy, cells were plated onto 35-mM FluoroDishes (World Precision Instruments) and pretreated with doxycycline (1.0 µg/ml), where indicated. Cells were grown in phenol-red free DMEM supplemented with 25 mM Hepes, pH 7.2, and 10% FBS and viewed in a custom-built heated chamber warmed to 37°C. Live cell images were obtained on a Nikon Eclipse TE2000U Inverted Microscope using an Eclipse EZ-C1 system (v. 3.90; Nikon) and a Plan Apo 60x Pan Apo (NA 1.40) objective lens. EGFP fluorescence was excited with the 488-nm line from a Melles Griot 488 Ion Laser and detected with a 515/30 emission filter; mRFP/cherry fluorescence was obtained using Melles Griot 543 laser excitation and a 590/50 emission filter. Z-series were collected every 1 min (20 optical sections with a step size of 0.8 µm). Z-series were volume- and time-rendered using C1 software (Nikon) and displayed as maximum z-projection AVI files. Gamma, brightness, and contrast were adjusted on the AVI files using Adobe Photoshop CS3, labels were added, and the files were rendered as MOV files.
6
Visualizing Cholesterol Dynamics in NSPCs
7
FCCP-Induced Mitochondrial Fission Assay
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Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) (Sigma) was used at a final concentration of 20 μM in full DMEM and was applied for 2 h at 37 °C to the cells, before they were fixed and stained. For live-cell imaging of FCCP induced fission, cells were seeded two days prior to the assay in FluoroDishes (World Precision Instruments) and treated for 45 min with 20 µM FCCP before imaging.
8
Imaging HCV Core Protein Trafficking
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Huh7.5 cells were infected with concentrated HCV TC-core at a multiplicity of infection of 1 for 24 h, transfected with AP-4–mCherry by using Lipofectamine 2000 (Invitrogen), and then seeded onto collagen-coated 35-mm FluoroDishes (World Precision Instruments, Inc.) (8 (link), 39 (link)). When specified, TC-core-infected cells were incubated with dimethyl sulfoxide (DMSO) or PIK93 (0.5 µM) for 3 h beginning at 72 h postinfection. At 72 h postinfection, cells were labeled with biarsenical dye and time-lapse images were taken and analyzed as described in Text S1 .
Generation of AAK1 and GAK knockout cell lines was performed as described inText S1 .
Generation of AAK1 and GAK knockout cell lines was performed as described in
9
Cytosolic pH Measurement in ciPTEC-T1 Cells
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ciPTEC-T1 were seeded in 35 mm Fluorodishes (World Precision Instruments GmbH) and cultured accordingly. Cells were loaded with the cytosolic pH-sensitive reporter molecule BCECF-AM (5 µM, 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxy methyl ester) (Thermo Fisher Scientific) in KHH buffer and incubated for 15 minutes at 37 °C. Cells were washed three times using KHH buffer and imaging was performed using an inverted microscope (Axiovert 200M, Carl Zeiss). BCECF fluorescence was sequentially excited at the isosbestic point (440 nm) (100 ms) and at the H+-sensitive wavelength (490 nm) (100 ms). Fluorescent images were captured at 530 nm emission wavelength. Images were analysed using Image Pro Plus software (version 6.3, Media Cybernetics). After background correction, the 490/440 emission ratio was used to quantify cellular pH45 (link), 51 (link).
10
Quantitative Mitochondrial Morphology Imaging
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Cells were seeded in 35 mm Fluorodishes (World Precision Instruments GmbH) and after exposure loaded with loaded with tetramethylrhodamine methyl ester (100nM) (TMRM, Thermo Fisher Scientific) for 25 minutes at 37 °C, 5% (v/v) CO2. Next, cells were washed twice using Krebs-Henseleit buffer supplemented with HEPES (10 mM, KHH) (pH 7.4), and images were captured using a temperature-controlled chamber connected to an inverted microscope (Axiovert 200M, Carl Zeiss) using a x63, 1.25 NA Plan NeoFluor oil immersion objective. As a positive control, the known uncoupling agent carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) was used at the end of each measurement. Images were corrected for background and uneven illumination followed by analysis where images were masked with a binarised image for mitochondrial morphology using Image Pro Plus software (version 6.3, Media Cybernetics) as previously described48 (link).
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