Ultraview vox 3d live cell imaging system

NPCs grown on coverslips coated with poly-D-lysine were infected with HCMV at an MOI of 0.5, and mock infection was performed as a control. Coverslips were collected at the indicated time points post infection and processed for IFA as described previously [83 (link)]. Mouse monoclonal antibodies for HCMV IE1 (IgG2a, clone p63-27, made in Prof. Britt’s lab) and Hes1 (IgG1, Abcam, Cat# ab119776) were used as primary antibodies, and FITC-anti-mouse-IgG2a (Invitrogen, Cat# 11-4210-82) and TRITC-anti-mouse-IgG1 (Southern Biotechnology, Cat# 1070–03) were applied as secondary antibodies. Nuclei were counterstained with Hoechst 33342 (Life Technologies). Images were obtained using a two-photon microscopy with the UltraVIEW VoX 3D Live Cell Imaging System (Perkin Elmer).

For the immunofluorescence analysis, liver tissues were fixed in 4% paraformaldehyde for 12 h at 4°C and then dehydrated in 30% sucrose solution. The tissues were then frozen in OCT (Sakura, Torrance, CA, United States) compound and sectioned into 20 μm slices using a freezing microtome (Leica, Germany). OCT was removed by washing three times in PBS, and the sections were immunostained with Alexa Fluor 647 anti-mouse F4/80 (BioLegend, Clone: BM8, Catalog: 123122), Alexa Fluor 647 anti-mouse CD11c (BioLegend, Clone: N418, Catalog: 117312) or Alexa Fluor 647 anti-mouse NK1.1 (BioLegend, Clone: PK136, Catalog: 108720) at 1:200 dilution. All the sections were imaged with Olympus IX83 confocal microscope outfitted with an UltraVIEW VoX 3D live cell imaging system (PerkinElmer). Images were analyzed with Image J software (National Institutes of Health).

NPCs grown on coverslips coated with poly-D-lysine were infected with ZIKV at an MOI of 3 or 0.1, and mock-infected NPCs were used as a control. Coverslips were collected at the indicated time points post-infection and processed for IFA as described previously (Liu et al., 2017 (link)). For cryosections, perfused tissues were fixed in 4% PFA, dehydrated in 30% sucrose, and frozen in tissue freezing medium (Leica Biosystems, Germany). Coronal sections (thickness of 40 μm) were used for immunofluorescence staining as described previously (Tang et al., 2015 (link)). Mouse monoclonal antibodies for DCX (Abcam, Cat#ab18723), Tbr1 (Abcam, Cat#ab31940), Ctip2 (Abcam, Cat#18465) and anti-ZIKV human serum (Ma et al., 2017 (link)) were used as primary antibodies. Secondary antibodies included FITC-anti-mouse-IgG2a (Invitrogen, Cat#11-4210-82), Alexa Fluor 488 goat-anti-human-IgG (Invitrogen, Cat#A-11013) and TRITC-anti-mouse-IgG1 (Southern Biotechnology, Cat#1070-03). Nuclei were counterstained with DAPI (Life Technologies). Images were obtained using a two-photon microscopy with the UltraVIEW VoX 3D Live Cell Imaging System (Perkin Elmer).

Transmission electron microscopy images were obtained from a JEM-2100 (JEOL) transmission electron microscope. FT-IR spectrum was measured on a Spectrum Two Fourier Transform Infrared (FT-IR) Spectrophotometer (Perkin-Elmer). XPS was recorded with an ESCALAB 250Xi photoelectron spectrometer (Thermo Fisher) using monochromatic Al Kα X-ray source. Powder X-ray diffraction (XRD) analysis was performed with a Rigaku MiniFlex 600 X-ray diffractometer using Cu-Kα (λ = 1.5418 Å). Zeta potential and particle size were studied by using a zeta sizer (Nano ZS, Malvern Instruments). Ultraviolet and visible diffuse reflectance spectroscopy (UV–Vis DRS) was measured with a Shimadzu UV-3600 UV–Vis spectrophotometer. Immunofluorescence sections were performed on a wide-field fluorescence microscopy (Olympus IX73). Spinning disk confocal microscopy images were obtained on a Perkinelmer UltraVIEW VoX 3D live cell imaging system. Small animal fluorescence imaging was performed with a Perkin-Elmer living image In Vivo Imaging System (IVIS) spectrum. Bioluminescence intensity and optical density were measured with a SpectraMax i3x multi-mode detection platform (Molecular Devices).

HepG2 and SMMC771 cells were labelled with GFP. Chol-let-7a and negative control mimics labelled with Cy5 were purchased from Ribobio (Guangzhou, China).
The GFP-labelled cells (2–3 × 10⁴) were seeded in 8-well BD Falcon™ and BD BioCoat™ Culture Slides (Becton Dickinson Labware, Franklin Lakes, NJ, USA). After 48 h, cells were transfected with Cy5-labelled Chol-let-7a or the negative control mimics (Chol-miRCtrl).
For live-cell imaging, cells were continuously observed using a PerkinElmer UltraVIEW VoX-3D Live Cell Imaging System (Shanghai, China) from 24 to 72 h post-transfection. Digital images were produced using Volocity Demo software (version 5.4, 32-bit). Co-localization events were calculated using the Volocity Demo software as described in the manufacturer’s recommendations. The experiment was repeated 3 times and all samples for each individual experiment were scanned at 5 different locations.
For electron microscopy, cells were collected at 48 h and 60 h after transfection and were fixed with 2.5% glutaraldehyde for 30 min at room temperature, followed by 1.5 h in 2% OsO₄. Samples were stained and examined with a transmission electron microscope (JEOL JEM 1010, Tokyo, Japan), and digital images were obtained with an Erlangshen ES1000W camera (Model 785, Gatan, Warrendale, PA, USA).

Measurement of the intracellular Ca²⁺ levels was performed in γδ T cells loaded with 2.5 μm fluo-4 AM (Invitrogen, USA) for 45 min at room temperature in HBSS. γδ T cells were washed, resuspended in HBSS and seeded on Lab-Tek^TM glass chamber slides (Nunc, Naperville, IL, USA) for 1 h at room temperature. When indicated, HA-his (3 μg/ml), HAF (3 μg/ml) and an anti-CD3 mAb (5 μg/ml) were added (time point: 0 min). Measurements of the intracellular Ca²⁺ responses were performed with an UltraVIEW VoX 3D Live Cell Imaging System (PerkinElmer, USA). γδ T cells were illuminated every 20 s and monitored for changes in intracellular Ca²⁺ levels by videomicroscopy for 1 h.

FHM cells were cultured in DMEM medium supplemented with 10% fetal bovine serum until reaching 80%–90% confluence. According to the manufacturer’ instructions, transfection agent of Fugene 6 (Promega, Shanghai, China) and plasmids were transfected into FHM cells at a ratio of 2:1 (v/v), before 4 μg of plasmid DNA was added to the medium in a 6-well plate. After 6 h of incubation, the medium was removed, and the cells were cultured in fresh medium at 28 °C with 5% CO₂. 36 h after transfection, cells were subsequently fixed with 4% (w/v) paraformaldehyde for 10 min, permeabilized with 0.1% (v/v) Triton X-100 for 10 min, and blocked the nonspecific binding with 3% (v/v) BSA at 37 °C for 1 h. The slides were washed three times with PBS and then incubated with mouse anti-HA antibodies (1:3000, ABclonal) and FITC-conjugated goat anti-mouse IgG (1:200, ABclonal) at 37 °C for 1 h, successively. The nuclei of all cells were stained with 1 mg/mL Hoechst 33,342 at room temperature for 10 min and photographed on UltraVIEW VoX 3D Live Cell Imaging System (PerkinElmer, Waltham, MA, USA). Cells transfected with pcDNA3.1 were used as negative control.