For laser injury assay cells cultured on coverslips were transferred in cell imaging media (CIM; HBSS with 10mM HEPES pH 7.4) or PBS (Sigma Aldrich, USA) into Tokai Hit microscopy stage top ZILCS incubator (Tokai Hit Co., Japan) maintained at 37°C. For laser injury, a 1-2μm-2 area was irradiated for <100 milliseconds with a pulsed laser (Ablate!, 3i Intelligent Imaging Innovations, Inc. Denver, CO, USA). To monitor ESCRT kinetics cells expressing appropriate tagged ESCRT protein was injured and imaged in spinning disc confocal or TIRF mode using IX81 Olympus microscope (Olympus America, PA) equipped with Yokogawa spinning disc confocal or Cell-TIRF (Olympus USA) system respectively. For all imaging a 60X-1.45NA oil objective was used. To quantify cell membrane repair, 1 ug/uL FM1-43 dye (Life technologies, USA) was added to the cell imaging buffer used and cells were imaged at 10 second intervals using IX81 Olympus microscope (Olympus America, PA). FM dye intensity was used to quantify the kinetics of cell membrane repair.
Ix81 microscope
Manufactured by Olympus
Sourced in Japan, United States, Germany, United Kingdom, Canada, Switzerland, Panama
The IX81 is an inverted research microscope designed for advanced fluorescence imaging applications. It features a sturdy frame, high-precision optics, and a range of accessories to support various experimental requirements. The IX81 provides a stable platform for delivering consistent, high-quality imaging results.
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Lab products found in correlation
Metamorph software, by Molecular Devices (13 mentions)
Hoechst 33342, by Thermo Fisher Scientific (12 mentions)
Matrigel, by BD (11 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (9 mentions)
Dp71 camera, by Olympus (8 mentions)
Orca er camera, by Hamamatsu Photonics (8 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (7 mentions)
Metamorph 7, by Molecular Devices (7 mentions)
Fluo 4 am, by Thermo Fisher Scientific (7 mentions)
Fura 2 am, by Thermo Fisher Scientific (7 mentions)
Triton x 100, by Merck Group (7 mentions)
Fm1 43 dye, by Thermo Fisher Scientific (6 mentions)
Microscopy stage top zilcs incubator, by Tokai Hit (6 mentions)
Fv1000 confocal laser scanning system, by Olympus (6 mentions)
Cellr software, by Olympus (6 mentions)
Em ccd camera, by Hamamatsu Photonics (6 mentions)
Lsm 710 confocal microscope, by Zeiss (6 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (5 mentions)
Matrigel, by Corning (5 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (5 mentions)
730 protocols using ix81 microscope
1
Laser-Induced Membrane Repair Assay
2
Laser-Induced Membrane Repair Assay
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For laser injury assay cells cultured on coverslips were transferred in cell imaging media (CIM; HBSS with 10mM HEPES pH 7.4) or PBS (Sigma Aldrich, USA) into Tokai Hit microscopy stage top ZILCS incubator (Tokai Hit Co., Japan) maintained at 37°C. For laser injury, a 1-2μm-2 area was irradiated for <100 milliseconds with a pulsed laser (Ablate!, 3i Intelligent Imaging Innovations, Inc. Denver, CO, USA). To monitor ESCRT kinetics cells expressing appropriate tagged ESCRT protein was injured and imaged in spinning disc confocal or TIRF mode using IX81 Olympus microscope (Olympus America, PA) equipped with Yokogawa spinning disc confocal or Cell-TIRF (Olympus USA) system respectively. For all imaging a 60X-1.45NA oil objective was used. To quantify cell membrane repair, 1 ug/uL FM1-43 dye (Life technologies, USA) was added to the cell imaging buffer used and cells were imaged at 10 second intervals using IX81 Olympus microscope (Olympus America, PA). FM dye intensity was used to quantify the kinetics of cell membrane repair.
3
Mitochondrial Function Evaluation in HepG2 Cells
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Mitochondrial function was evaluated via detecting the mitochondrial membrane potential and measuring the ROS levels. HepG2 cells were incubated with the JC-1 probe for 30 min at 37 °C in the dark [32 (link)]. Subsequently, cells were washed with PBS to remove the free JC-1 probe. Then, nuclei were stained with DAPI and the mitochondrial potential was assessed under an Olympus IX81 microscope using FV10-ASW 1.7 software. The ImageJ software was used to analyze the mitochondrial potential as described previously [33 (link)]. Cellular ROS measurements were performed using the DHE probe. HepG2 cell was incubated with 5 μM DHE for 30 min at 37 °C in the dark. Then, cells were washed with PBS to remove free ROS probe. Subsequently, cellular ROS was observed under the Olympus IX81 microscope and quantified by fluorescence activated cell sorting (FACS) [34 (link)].
4
Competitive BiFC of PNPLA3 interactions
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COS-7 cells were transfected in 12-well plates with coverslips using Lipofectamine LTX (ThermoFisher) with 100 ng mCherry tracer to identify transfected cells, 500 ng Yn-ABHD5, and either 500 ng WT PNPLA3-Yc, PNPLA3 I148M-Yc, PLIN5-Yc, or bFOS-Yc as specified in the figure legend. Cells were incubated at 30°C with 200 μM OA overnight to promote lipid droplet formation. The next day, the cells were fixed with 4% paraformaldehyde before being imaged in phosphate buffered saline (PBS). Reconstituted EYFP signal in mCherry+ cells were acquired using an Olympus IX-81 microscope equipped with a spinning disc confocal unit. Competitive BiFC20 (link) was performed in COS-7 cells seeded in 12-well plates with coverslips. Cells were co-transfected with 400 ng Yn-ABHD5 and 400 ng PNPLA2-Yc, and either 200 ng mCherry, PNPLA3-mCherry, PNPLA3 I148M-mCherry, or Plin1-mCherry (4:4:2 ratio of Yn:Yc:mCherry). Cells were incubated with 400 μM OA overnight to promote lipid droplet formation. Reconstituted EYFP signal in mCherry+ cells were acquired using an Olympus IX-81 microscope equipped with a spinning disc confocal unit in a blind manner. Five random 40× fields were captured for each coverslip, and cells were scored for the presence of EYFP fluorescence in mCherry+ cells by an analyst who was blinded to the experimental conditions.
5
Time-Lapse Confocal Imaging of Zebrafish Neuromasts
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We performed time-lapse confocal fluorescence microscopy for up to 40 hrs on live animals. Embryos of 2–5 dpf were anesthetized in 600 μM 3-aminobenzoic acid ethyl ester methanesulfonate in E3 medium and mounted in a 35 mm glass-bottomed dish in 1 % low-melting-point agarose.
Transgenic larvae expressing actb1-EGFP were imaged under a 100X/NA 1.35 silicone-oil objective lens on an Olympus IX81 microscope equipped with a microlens-based, super-resolution confocal system (VT iSIM, VisiTech International). Neuromasts were imaged at intervals of 5 min as Z-stacks acquired with 0.5 μm steps under laser excitation at 488 nm. To keep the regions of interest within view over the imaging period, we developed a customized tracking algorithm that automatically updated the stage position, correcting for sample drift. The code is available athttps://github.com/a-jacobo/AutoTracker .
Transgenic larvae expressing lynEGFP were imaged under a 60X/NA 1.3 silicone-oil objective lens with an Olympus IX81 microscope equipped with a spinning-disk confocal system (Ultraview, Perkin-Elmer, Waltham, MA). Neuromasts were imaged at intervals of 5 min as Z-stacks acquired with 0.5 μm steps under laser excitation at 488 nm.
Transgenic larvae expressing actb1-EGFP were imaged under a 100X/NA 1.35 silicone-oil objective lens on an Olympus IX81 microscope equipped with a microlens-based, super-resolution confocal system (VT iSIM, VisiTech International). Neuromasts were imaged at intervals of 5 min as Z-stacks acquired with 0.5 μm steps under laser excitation at 488 nm. To keep the regions of interest within view over the imaging period, we developed a customized tracking algorithm that automatically updated the stage position, correcting for sample drift. The code is available at
Transgenic larvae expressing lynEGFP were imaged under a 60X/NA 1.3 silicone-oil objective lens with an Olympus IX81 microscope equipped with a spinning-disk confocal system (Ultraview, Perkin-Elmer, Waltham, MA). Neuromasts were imaged at intervals of 5 min as Z-stacks acquired with 0.5 μm steps under laser excitation at 488 nm.
6
Competitive BiFC of PNPLA3 interactions
7
Quantifying Apoptosis in Neural Stem Cells
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The apoptosis of NSCs in the brain tissue was detected using TUNEL (TdT-mediated dUTP nick-end labeling) staining for the DNA fragmentation of NSCs with the TUNEL fluorescence kit (G2350, Promega). After dewaxing, hydration, immersion in 4% PFA for 15 min, and permeabilization with 20 μg/mL proteinase K solution for 10 min, 50 μL TUNEL solution was added to each slide and placed at 37 °C for 1 h. The reaction was terminated with 2× SSC (sodium citrate salt) for 15 min, and the sections were stained with DAPI. Finally, the green fluorescence (fluorescein-12-dUTP) and apoptotic cells’ blue background (DAPI) were examined under an Olympus IX81 microscope and analyzed using Image J software.
In the in vitro experiments, NSCs derived from the hippocampus and striatum tissues of the rat offspring and inoculated on coverslips after 7 days of intervention were fixed with 4% PFA for 20 min, permeabilized with 0.3% Triton X-100 for 20 min and incubated with TUNEL reaction mixture at 37 °C for 1 h. The reaction was terminated with 2× SSC for 15 min, and the cells were stained with DAPI. TUNEL-positive NSCs were identified using an Olympus IX81 microscope and analyzed with Image J software.
In the in vitro experiments, NSCs derived from the hippocampus and striatum tissues of the rat offspring and inoculated on coverslips after 7 days of intervention were fixed with 4% PFA for 20 min, permeabilized with 0.3% Triton X-100 for 20 min and incubated with TUNEL reaction mixture at 37 °C for 1 h. The reaction was terminated with 2× SSC for 15 min, and the cells were stained with DAPI. TUNEL-positive NSCs were identified using an Olympus IX81 microscope and analyzed with Image J software.
8
Multilineage Differentiation Assay of Adipose-Derived Stem Cells
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Human Adipose-Derived Stem Cells (ADSC) were purchased from Invitrogen (Invitrogen, Grand Island, NY, Cat. No. R7788115) and ZenBio (ZenBio Inc, Research Triangle Park, NC, Cat. No. ASC-F). All donors were non-diabetic females. Cells were maintained in Complete MesenPRO RS™ Medium (Invitrogen). Chondrogenesis was induced using STEMPRO® Chondrogenesis Differentiation Kit according to manufacturer’s protocol (Invitrogen). After 21 days, cells were fixed, stained with Alcian Blue for proteoglycan content and phase contrast images were taken using an Olympus Ix81 microscope. Osteogenesis was induced using the STEMPRO® Osteogenesis Differentiation Kit (Invitrogen) for 21 days and assessed using Alizarin Red staining to visualize calcium depositions in extracellular matrix. Adipogenesis was induced with STEMPRO® Adipogenesis Differentiation Kit (Invitrogen) for 14 days and assessed using Oil-Red-O staining. Images were acquired at 4x and 10x magnification (Olympus Ix81 microscope).
9
Quantifying Cell Invasion and Colony Formation
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Colony formation assays in soft agar were performed in triplicate as described earlier [43 (link)]. Images were captured by Olympus IX81 microscope using Image-Pro Plus software (Olympus) at 100× optical magnification and colony-forming efficiency was quantified. Matrigel invasion assay was performed as described previously [44 (link)]. Images were captured by Olympus IX81 microscope at 40X magnification. Statistical analysis was performed by Student’s t test using GraphPad software with level of significance P <0.001 (GraphPad software, San Diego, CA, USA).
10
Imaging Cellular Dynamics with Microscopy
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Long-term imaging was done using an Olympus IX81 microscope equipped with temperature, humidity, and CO2 control (Olympus, Tokyo, Japan) or a biostation (Nikon, Melville, NY). Acquisitions were typically obtained over a period from 12 to 24 h using 10× or 20× objectives. TIRF images were obtained through a 60× oil immersion objective with an iLas2 TIRF system connected to an Olympus IX81 microscope equipped with temperature, humidity, and CO2 control. Confocal images of phalloidin, phospho-myosin, GFP-mDia2, and FHOD3 were obtained using an LSM710 confocal microscope (Zeiss, Jena, Germany) with a 63× water objective.
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