To stain proteins for immunofluorescence microscopy, cells were fixed with 4% formaldehyde and permeabilized in permeabilization buffer (PS; 0.05% saponin, 1% BSA, and 0.05% NaN3 in PBS) for 20 min. Samples were incubated with rabbit anti-ASC (1:200 in PS) for 2 h, washed with PBS, and subsequently incubated with Alexa Fluor 594–coupled goat anti–rabbit IgG (1:1,000), Hoechst 33342 (1:5,000), and, where indicated, with Alexa Fluor 647–coupled VHHASC or VHH NP1 (1 µg/ml) for 1 h. Samples were washed with PBS and H2O, and mounted with Fluoromount-G (Southern Biotech) or Duolink In Situ Mounting Medium with DAPI (Sigma-Aldrich). Images were acquired using a PerkinElmer Ultraview Spinning Disk Confocal microscope or a Nikon Eclipse Ti wide field microscope. ASC foci and nuclei in wide field microscopy images were quantified using the spot detection function of the Imaris software package (Bitplane).
Eclipse ti widefield microscope
Manufactured by Nikon
The Eclipse Ti widefield microscope is a high-performance optical microscope designed for a variety of laboratory applications. It features a large working distance, high numerical aperture objectives, and a wide field of view for efficient sample observation and analysis.
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6 protocols using eclipse ti widefield microscope
1
Immunofluorescence Imaging of ASC Inflammasome
2
Live-cell Imaging of Dengue and SARS-CoV-2
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Huh7-Lunet-T7 cells expressing the dengue reporter constructs (Lunet-T7-RC) were seeded onto a glass bottom 33 cm2 dish (Mattek) at a density of 2 × 104. Transfection of the pIRO-D system (28 (link)) was performed at 24 h postseeding using the TransIT-LT1 (Mirus) transfection reagent according to the manufacturer’s instructions. Four hours posttransfection (hpt) the transfection medium was exchanged for complete medium lacking phenol red (imaging medium). Images were collected with a Perkin Elmer spinning disk confocal microscope. For SARS-CoV-2 live cell imaging, A549-ACE2 or a selected clone of A549-ACE2 stably expressing the fluorescent reporter (A549-ACE2-RC), were seeded on 35-mm Ibidi dishes with gas permeable membrane and sealable lid. Cells were infected for 1 h with SARS-CoV-2 (MOI = 10) and at 2 hpi the medium was exchanged for imaging medium. The lid was moved to the locked position and silicon was used to seal the dish in order to prevent evaporation. Images were collected with a Nikon Eclipse Ti widefield microscope. Multiple observation fields were imaged for 8 h or 18 h at an interval of 10 min for transfection or infection, respectively.
3
Live Cell Imaging of ULK1/2 DKO Cells
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For live cell imaging, MEF ULK1/2 DKO cells expressing Flag-ULK1, mCherry-DFCP1, and GFP-LC3 were plated onto a four compartment 35-mm glass bottom dish (Ibidi) 24 h before imaging. On the day of imaging, the cells were washed twice with pre-warmed EBSS followed by treatment with EBSS or EBSS and 2 μM MRT68921 and were immediately transferred to the microscope stage. Two to three fields were picked and imaged every 1 min for a total of 60 min using a Nikon Eclipse Ti wide-field microscope.
4
Automated Quantification of Mitotic Cells
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The Eclipse Ti wide‐field microscope (Nikon) with a Plan Fluor 40× NA 1.3 objective was used to capture fixed cells. Optical sections were acquired every 0.7 μm using the ORCA‐Flash 4.0 CMOS camera C11440‐22CU (Hamamatsu). The numbers of histone H3S10ph‐positive nuclei in G1 cells were determined by an automated pipeline using the software CellProfiler (Kamentsky et al, 2011 ). The 3D data sets were projected (maximum intensity projection) and converted into TIFF files using standard CellProfiler modules. The analysis was performed using the following modules: IdentifyPrimaryObjects, MeasureObjectIntensity, ClassifyObjects and FilterObjects. The nuclei were identified using Hoechst 33342 staining. Transfected cells were identified based on the EYFP signal. Among the cyclin A2‐negative nuclei (Alexa 647 fluorescence signal), the cells positive for histone H3S10ph were identified using the Alexa 594 signal. Detailed pipeline description is available upon request from Jan Ruppert.
5
Cell Cycle Phase Dynamics Imaging
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Cell cycle phase lengths were assessed using a PCNA-RFP intra-chromosomal reporter (Muramoto and Chubb, 2008 (link)). Cells were seeded at 1x104 cells/ml in filter-sterilised HL5 with or without 75mM glucose and grown for 48 hours (growing to 3-5x106 cells/ml). Cells were then diluted to 1x105 cells/ml in G+ or G- HL5, added into each well of a glass-bottomed multi-chamber slide (Ibidi), and left to settle for 2 hours before imaging every 4 minutes for 16 hours using an Eclipse Ti widefield microscope (Nikon) with laser-assisted auto-focus, with illumination provided by a Precise LED light source at 25-50% maximum intensity. Images were subsequently analysed manually using NIS Elements Viewer (Nikon).
6
Microglia Dynamics Imaging and Tracking
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Microglia cultured in 96‐well plates were labeled with Hoechst (1 μg/ml) in growth media for 45 min, washed with serum‐free media and incubated in Opti‐MEM (containing vehicle or drugs as stated) for 15 min prior to imaging. Cells were imaged on a Nikon Eclipse Ti widefield microscope with a stage incubator maintaining 37°C/5% CO2 using both phase contrast and DAPI channels with low UV laser power to prevent toxicity. Images were obtained every 5 min for 3 h.
The fluorescent (nuclear) channel was then analyzed for cell movement in ImageJ by first performing a rolling‐ball background subtraction (ball radius 20) and loading the resulting images into the TrackMate plugin (Tinevez et al., 2017 (link)). To identify nuclei, LoG detection was used with a spot size of 8 μm, thresholds were empirically determined for each experiment and applied equally across all conditions. No spot filtering was necessary. Tracking was performed using the simple LAP tracker with max linking distance of 40 μm, and max gap closing distance of 70 μm over two frames.
The fluorescent (nuclear) channel was then analyzed for cell movement in ImageJ by first performing a rolling‐ball background subtraction (ball radius 20) and loading the resulting images into the TrackMate plugin (Tinevez et al., 2017 (link)). To identify nuclei, LoG detection was used with a spot size of 8 μm, thresholds were empirically determined for each experiment and applied equally across all conditions. No spot filtering was necessary. Tracking was performed using the simple LAP tracker with max linking distance of 40 μm, and max gap closing distance of 70 μm over two frames.
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