Wild-type, Ire1 knockout and Perk knockout MEFs were gifts from Dr. David Ron (University of Cambridge). Atf6 knockout MEFs were provided by Dr. Randal Kaufman (UC San Diego). Bax/Bak double knockout and Bax/Bak double knockout rescued by wild-type Bak MEFs were gifts from Dr. Navdeep Chandel (Northwestern University). Xbp1+/+ and Xbp1−/− MEFs were gifts from Dr. Laurie Glimcher (Cornell University). These MEFs were cultured in DMEM supplemented with 10% FBS DMEM and antibiotics. HEK293 cells were cultured in DMEM supplemented with 10% FBS DMEM and antibiotics. For live-cell imaging, HEK293 or Ire1α knockout MEF cells were plated on a poly-L-lysine coated glass-bottom 35 mm dish (Mattek, Ashland, MA) the day before the analysis. The cells were then cultured with 10% FBS-DMEM containing 1 or 5 μg/ml tunicamycin in association with 0.5 μg/ml of PI or 100 nM tetramethylrhodamine methyl ester (TMRM) in 5% CO2 at 37°C for 12–24 h. The images were captured every 20 min using Yokogawa spinning disk confocal on a Nikon TE-2000E2 inverted microscope with a 40x objective lens. Analyses were done on 16 bit or 32 bit images using ImageJ 1.45.
Te2000 e2 inverted microscope
Manufactured by Nikon
The Nikon TE2000-E2 is an inverted microscope designed for a wide range of applications. It features a stable, ergonomic design and advanced optical components to provide high-quality imaging. The TE2000-E2 is capable of various microscopy techniques, including phase contrast, fluorescence, and brightfield imaging, making it a versatile tool for research and analysis.
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Lab products found in correlation
Metamorph 7, by Molecular Devices (2 mentions)
Bodipy 493 503, by Thermo Fisher Scientific (1 mentions)
Prolong gold with dapi, by Thermo Fisher Scientific (1 mentions)
Microscope stage top incubation chamber, by Tokai Hit (1 mentions)
Celltracker green, by Thermo Fisher Scientific (1 mentions)
Gfr matrigel, by Corning (1 mentions)
Rat anti mouse f4 80 primary antibody, by Bio-Rad (1 mentions)
Prolong gold antifade with 4 6 diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions)
Csu 10, by Nikon (1 mentions)
Insulin, by Agilent Technologies (1 mentions)
Foxo1, by Cell Signaling Technology (1 mentions)
Perfect focus system, by Nikon (1 mentions)
9 protocols using te2000 e2 inverted microscope
1
Live-cell imaging of ER stress in MEFs
2
Confocal Imaging of Fluorescent Particles
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Immunofluorescence Staining of Hig2 Protein
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Cells were fixed in 10% buffered formalin in PBS for 1 h, blocked in 1% normal goat serum in PBS for 1 h at room temperature, incubated with Hig2 antibodies (1:100, Rockland Immunochemicals [24] (link)) for 2 h at room temperature, incubated with fluorescent secondary antibodies 1:1000 for 1 h, treated with Bodipy 493/503 (ThermoFisher, D-3922) at 1:10,000 for 15 min, and mounted with Prolong Gold with DAPI (Life Technologies). Cells were imaged at room temperature with a Solamere Technology Group modified Yokogawa CSU10 Spinning Disk Confocal with a Nikon TE-2000E2 inverted microscope at 60×.
4
Live Imaging of Pericyte-Endothelial Cell Interactions
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Sorted pericytes (CD146+/CD90+/CD56−/CD45−/CD34−/CD31−) (passage <8) were maintained in basal growth medium and kept at <70% confluence. Cells were then seeded at a density of 1.2 × 104 cells/cm2 on growth factor reduced (GFR)-Matrigel (Corning)-coated 24-well plates in endothelial growth medium on GFR-Matrigel. Pericytes were also separately seeded on Matrigel substrates in co-culture in a 1:10 ratio with primary human pulmonary artery endothelial cells (hPAECs, P8) (Lonza). For co-culture experiments, populations were labeled with Cell Tracker Green (pericytes) or Red (hPAECs) (Invitrogen). Cells were cultured at 37°C and 5% CO2 in a humidified microscope stage-top incubation chamber (Tokai Hit). Cells on Matrigel were visualized using phase-contrast and epifluorescence microscopy on a Nikon TE-2000-E2 inverted microscope equipped with FITC and tetramethylrhodamine isothiocyanate filter cubes and a 10× objective, and images were captured every 10 min for up to 50 hr using a CoolSNAP ES2 Monochrome 1,394 × 1,040 High Resolution Camera and NIS Elements Software 3.2. Images were also captured after 1, 3, and 5 days of culture.
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Confocal Microscopy of Thrombi
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Cellular Uptake and Localization of Functionalized Particles
7
Immunostaining of Mouse Islet Cells
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Dispersed mouse islets were fixed in 4% PFA for 10 minutes and stored in PBS at 4°C. Fixed cells on coverslips were immunostained as described (15 (link)). Briefly, coverslips were blocked in goat serum–based block plus 0.1% Triton-X, then stained with the following antibodies: Insulin (1:200; Dako) and FOXO1 (1:50; Cell Signaling Technology), followed by Alexa Fluor secondary antisera (Invitrogen), and coverslips were mounted onto slides using FluoroshieldÔ with Dapi (Sigma-Aldrich). Coverslips were imaged on either a Leica SP-5 Laser Scanning Confocal Fluorescence microscope, TissueGnostics Fluorescence Slide Scanner, or a Nikon TE2000-E2 inverted microscope. Quantification of nuclear FOXO1 was performed by a blinded individual utilizing a trinary scoring system.
8
Live Cell Imaging Protocols
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Live cell imaging was performed at room temperature in L15-ASW on a Nikon TE2000 E2 inverted microscope (Nikon, Melville, NY) with 60x oil immersion objective lens, with additional 1.5x magnification. Focus during long time-lapse sequences was maintained with the Nikon Perfect Focus system. Images were collected with a Cascade II charge-coupled device camera (Photometrics, Tucson, AZ) controlled by MetaMorph 7.8 software (Molecular Devices, Sunnyvale, CA). Imaging chamber was assembled as previously reported for cells cultured on coverslip [41 (link)], and fresh L15-ASW was supplemented for imaging longer than 1 h. For cells cultured on glass-bottom dish, fresh medium was exchanged every hour. Time-lapse series were acquired at 30 s intervals for 3.5 h (Figure 4(a) ); 60 s intervals for 3 h (Figure 4(b) ); 120 s intervals for 13 h (Figure 5(a) ); and 30 s intervals for 3 h (Figure 6(g) ).
9
Live-cell Imaging of Subcellular Dynamics
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Coverslips with cells were assembled into imaging chambers as described previously (Lee et al., 2008 (link)). Samples were imaged on Nikon TE2000 E2 inverted microscope (Nikon, Melville, NY) with 60× oil-immersion objective plus additional 1.5× magnification. X-cite 120 metal halide lamp (EXFO, Quebec City, QC, Canada) and corresponding filter sets (Chroma Technology, Bellows Falls, VT) were used for fluorescence imaging. Digital acquisition was supported by an Andor iXon 888 Ultra electron-multiplying charge-coupled device camera under the control of MetaMorph 7.8 software (Molecular Devices, Sunnyvale, CA). DIC time-lapse imaging was carried out at 5-s intervals.
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