Cells were plated over gelatin‐coated cell culture dishes and treated with CellROX Deep Red reagent (ThermoFisher Scientific) diluted in culture medium at a final concentration of 5 μM for 30 min. For image acquisition, cells were washed three times with PBS, stained with Hoechst 33342 diluted in culture medium and imaged using a 60× 1.4 NA Plan‐Apochromat VC objective on a Nikon Ti‐E equipped with a Yokogawa CSU‐X1 spinning disc head and a Photometrics Evolve 512 EMCCD camera. For flow cytometry analysis, cells were washed three times in PBS, trypsinized, resuspended in 0.5% BSA PBS and analysed using a FACS Aria II.
Plan apochromat vc
Manufactured by Nikon
Sourced in United Kingdom
The Plan Apochromat VC is a high-quality optical lens designed for use in laboratory and research equipment. It features an apochromatic design, which helps to minimize chromatic aberration, and a vibration compensation (VC) system to provide stable and clear images. The lens is optimized for a wide range of applications, including microscopy and imaging tasks.
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Lab products found in correlation
Ultraview vox, by PerkinElmer (3 mentions)
Eclipse te2000, by Nikon (3 mentions)
Volocity 6, by PerkinElmer (3 mentions)
A1 confocal microscope, by Nikon (3 mentions)
Plan apochromat, by Nikon (2 mentions)
A1r laser scanning confocal microscope, by Nikon (2 mentions)
Nis elements microscope imaging software, by Nikon (2 mentions)
Cellrox deep red reagent, by Thermo Fisher Scientific (1 mentions)
Biorevo microscope, by Keyence (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Plan apochromat 1.40 oil dic m27, by Zeiss (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Nis elements software, by Nikon (1 mentions)
11 protocols using plan apochromat vc
1
Oxidative Stress Measurement via CellROX
2
Live-cell Imaging with Spinning Disk Confocal Microscopy
3
Immunofluorescence Staining of Adherent Cells
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The cells were grown on a poly-l -lysine–coated coverslip. For immunostaining, the cells were fixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS; 140 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, and 8.1 mM Na2HPO4) for 15 min and permeabilized with 0.2% Triton X-100 in PBS for 5 min. The fixed cells were blocked with 3% bovine serum albumin (BSA)/PBS for 1 hour at room temperature. Then, the cells were incubated with the primary antibodies diluted with 3% BSA/PBS for 1 hour in a humidity box, stained with Alexa Fluor–conjugated secondary antibodies for 1 hour, counterstained with DAPI for 5 min, and mounted with VECTASHIELD (Vector Laboratories). Images were acquired using a BioRevo microscope (BZ-9000; Keyence) equipped with a 40× objective lens [Plan Apochromat, numerical aperture (NA) 0.95, Nikon] and a 100× oil objective lens (Plan Apochromat VC, NA 1.4). Cellular counting and image cropping were performed using ImageJ.
4
TBEV Immunofluorescence Assay in A549 Cells
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The first passage of rescued viruses was used to infect A549 cells for an immunofluorescence assay. A total of 4 × 104 cells grown on coverslips in 24-well plates were incubated with TBEV Torö-6A or Torö-38A at a MOI of 1 for 1 h at 37 °C in 100 μl serum-free DMEM. The virus inoculum was removed, cells were washed with PBS, 1 ml of DMEM-2% FCS was added, and the incubation was continued at 37 °C for 24 h. Cells were fixed with 3% paraformaldehyde (PFA), quenched with 10 mM glycine, and permeabilized with PBS and 0.5% Triton X-100 followed by incubation with primary antibodies against the E protein (mouse monoclonal 1786,3, diluted 1:50059 (link) and secondary Alexa 488 donkey anti-mouse IgG antibodies (Invitrogen, diluted 1:250)). Cells were incubated with DAPI (diluted 1:5000) before the coverslips were mounted and analysed using a Nikon A1R Laser Scanning Confocal Microscope (Nikon) with a 60 × oil immersion lens (Plan Apochromat VC) and NIS-Elements microscope-imaging software (Nikon).
5
Confocal Microscopy Immunofluorescence Staining
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The immunofluorescence staining was performed as described previously (Hirata et al., 2008 (link)). The samples were viewed with a confocal microscope (A1R, Nikon) equipped with oil-immersion (NA 1.40, 100×; Plan Apochromat VC, Nikon) and air (NA 0.95, 40×; Plan Apochromat, Nikon) objectives. Acquired images were analyzed off-line with the public domain ImageJ program (NIH).
6
Visualizing Golgi-ER Membrane Dynamics
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Cells were grown on coverslips to 20 to 40% confluence, transfected, and incubated for 24 h. Cells were washed, fixed, and stained with antibodies. Confocal images were acquired using a Nikon A1R laser scanning confocal microscope (Nikon) with a 60× oil immersion lens (Plan-Apochromat VC) under the control of NIS-Elements microscope imaging software (Nikon). For live-cell microscopy, 140,000 HeLa cells were seeded in a 35-mm MatTek glass-bottom dish. Cells were transiently transfected with eGFP-GBF1 and mCherry-viperin using Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations. After 8 h, the transfection medium was replaced with fresh medium, followed by incubation for another 16 h. Spinning-disc confocal live-cell microscopy was performed at 5% CO2 using a 63× objective lens (Plan-Apochromat 1.40 Oil DIC M27) in a Cell Observer spinning disc confocal microscope system (Andor iXon Ultra; Zeiss) controlled by ZEN software. Image analysis and preparation were completed using ImageJ and Adobe Photoshop CS5.
7
Confocal Microscopy for Protein Co-Localization
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Experiments were performed using a Nikon A1 confocal microscope equipped with a 60× oil immersion objective (Plan Apochromat VC, NA 1.4). For the co-localization experiments the pinhole size was set to 1 Airy unit (< 0.8 μm) and images with 1.5× zoom of 1024 × 1024 pixels were acquired. For the MKL2 translocation experiments, the pinhole size was set to 1 Airy unit (< 0.8 μm) and images were acquired with 1× zoom using tile scans, resulting in images of 4660 × 4660 pixels. Samples were excited with 447 nm, 514 nm and a 561 nm laser line, and reflected onto the sample by a 457/514/561 dichroic mirror. CFP emission was filtered through a BP482/35 emission filter; YFP emission was filtered through a BP540/30 emission filter; RFP emission was filtered through a BP595/50 emission filter. All acquisitions were corrected background signal. To avoid bleed-through, images were acquired with sequential line scanning modus.
8
Live-cell Imaging of Protein Dynamics
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Just before live-cell imaging, the medium of cells grown on MatTek dishes was changed to α-MEM without phenol red, supplemented with 20 mM HEPES, pH 7.4 and 5% FBS and placed into a temperature controlled chamber on the microscope stage with 95% air: 5% CO2 and 100% humidity. Live-cell imaging data were acquired using a fully motorized inverted microscope (Eclipse TE-2000, Nikon) equipped with a CSU-X1 spinning disk confocal head (UltraVIEW VoX, Perkin-Elmer) using a 60× lens (Plan Apochromat VC, 1.4 NA, Nikon) under control of Volocity 6.0 (Improvision, Perkin-Elmer). 14-bit digital images were obtained with a cooled EMCCD camera (9100-02, Hamamatsu, Japan). Four 50 mW solid-state lasers (405, 488, 561, and 647 nm; Crystal Laser and Melles Griots) coupled to individual acoustic-optical tunable filter (AOTF) were used as light source to excite EGFP and TagRFP-T. Rapid two-colour time-lapses were acquired at 500 ms to 2 s intervals, using a dual (525/50; 640/120, Chroma) emission filter, respectively. The power of the lasers supported excitation times of 50 ms in each wavelength and the AOTFs allowed minimum delay (~1 ms) between two colors (e.g., delay between green-red for each timepoint), which was an important factor to assess the colocalization between markers.
9
Visualizing Gα13 and G13 Sensor in HeLa Cells
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To obtain confocal images of live HeLa cells transiently expressing either a tagged Gα13 variant or G13 single plasmid sensor, a Nikon A1 confocal microscope, equipped with a 60x oil immersion objective (Plan Apochromat VC, NA 1.4), was used. The pinhole size was set to 1 Airy unit. To check the localization of tagged Gα13 variants, samples were excited with a 457nm laser line, a 457/514 dichroic mirror was used and the emission was filtered through a 482/35BP filter. To check the localization of the G13 single plasmid sensor constructs, samples were excited with a 440nm (CFP) and a 514nm (YFP) laser line, a 457/514 dichroic mirror was used and the emission was filtered through a 482/35BP (CFP) or 540/30BP (YFP), respectively. Images were acquired with sequential line scanning modus, to avoid bleedthrough.
10
Immunofluorescence Imaging of Transfected HUVECs
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Transfected HUVECs were grown to a monolayer, washed in PBS (1mM CaCl2 and 0.5 mM MgCl2) and fixed in a PBS solution (1mM CaCl2 and 0.5 mM MgCl2) with 4% formaldehyde. After fixation, HUVECs were permeabilized for 5 min in PBS containing 0.5% Triton X-100 and blocked for 20 min in PBS containing 0.5% Bovine serum albumin (BSA). Finally, HUVECs were incubated for 1 h with directly labelled antibodies, dissolved in 0.5% PBS-BSA. Confocal images were obtained on a Nikon A1 confocal microscope, equipped with a 60x oil-immersion objective (NA 1.40, Plan Apochromat VC) and Nikon NIS elements software.
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