ColorfulCell reporter plasmid (AddGene,#62449) expresses six fluorescent proteins which allow to visualize cell membrane (Cerulean), nucleus (TagBFP), Golgi apparatus (Citrine), mitochondria (AzamiGreen), endoplasmic reticulum (mCherry) and peroxisomes (iRFP670) [30 (link)]. WT and ISG20L2 J77 clones were electroporated with ColorfulCell plasmid as previously indicated in ‘cell transfection’ and incubated overnight. Glass plates (No. 1.5 Mat-Tek Corporation) were coated (1 h at 37 °C or overnight at 4ºC) with antibodies αCD3 (HIT3a, 4.87 μg/mL) and αCD28 (10 μg/mL) diluted in bicarbonate buffer (0.1 M NaHCO3, 0.32 M Na2CO3). Cells were seed onto glass plates covered with HBSS 1.5% FBS 25 mM HEPES. Live imaging was performed (at 37 °C, 5% CO2) on a Leica SP8 Navigator confocal laser unit (63 × oil objective). Images were analysed using Imaris Cell Imaging Software (Oxford Instruments).
Imaris cell imaging software
Manufactured by Oxford Instruments
Sourced in Switzerland
Imaris Cell Imaging Software is a comprehensive scientific imaging software platform designed to visualize, analyze, and quantify data from a variety of microscopy techniques. The software provides tools for 3D and 4D image reconstruction, object identification, tracking, and measurements, enabling researchers to extract meaningful insights from their cell imaging data.
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Lab products found in correlation
Lsm 710 meta confocal microscope, by Zeiss (1 mentions)
Lsm 980, by Zeiss (1 mentions)
Rabbit anti h3 cit antibody, by Abcam (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Gsk1016790a, by Cayman Chemical (1 mentions)
Anti rabbit igg af488, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Las x software, by Leica (1 mentions)
Remote manager version 3, by Scientific Volume Imaging (1 mentions)
5 protocols using imaris cell imaging software
1
Multicolor Cellular Visualization Assay
2
Immunofluorescence staining of tissue samples
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Tissues were fixed in paraformaldehyde (4% w/v in 0.1 mol L−1 PBS) for 45 min at room temperature (RT). After fixation, tissues were washed thoroughly for 1 h in PBS (0.01 mol L−1, pH 7.2 at RT). Tissues were blocked for 1 h (0.1 mol L−1 PBS containing 1% Triton X-100, 10% sheep serum). Tissues were incubated in primary antibody (diluted in 0.1 mol L−1 PBS containing 1% Triton X-100, 10% sheep serum, Supplementary Table 1 ) for 16 h at 4 °C and immunoreactivity was detected using the secondary antibodies listed in Supplementary Table 2 (1:500 in 0.1 mol L−1 PBS, 1 h at RT). Before mounting, tissues were washed thoroughly in PBS (0.1 mol L−1 PBS for 2 h at RT). Control tissues were prepared by omitting primary or secondary antibodies. Tissues were examined using as LSM710 Meta confocal microscope (Zeiss, Munich, Germany). Confocal micrographs were digital composites of Z-series scans (0.5–1 μm optical sections). Each antibody label was assessed in a minimum of three independent replicates to determine reproducibility and consistency. Final representative images were constructed using FIJI software19 (link). Depth-coding of confocal metafiles was performed using Imaris Cell Imaging Software (Oxford Instruments, Zurich, Switzerland).
3
Super-Resolution Imaging of Cell Volume
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Cells were seeded and stained as mentioned in the fluorescence imaging section. Full Z‐stacks of entire cells in isosmotic imaging media or hyperosmotic imaging media were acquired using an LSM 980 (Zeiss Instruments) microscope with ZEN 3.1 Blue software (Zeiss Instruments) in airyscan to perform super resolution imaging. Individual cell volumes of stressed and non‐stressed cells of all the variants were measured using the Imaris Cell Imaging Software (Oxford Instruments). Images from three independent experiments were analyzed and volumes of at least 20 cells per construct and condition were measured for a total of 213 cells. To obtain the relative volume change, the cell volumes of stressed cells were normalized to the non‐stressed cell volumes, and the difference was plotted as % of cell reduction.
4
Neutrophil NET Formation Quantification
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Neutrophils were seeded onto poly-L-lysine-coated coverslips in a 12-well plate at a density of 1 to 2×106 cells per well in culture medium. Cells were treated with 1 nmol/L, 10 nmol/L, or 100 nmol/L of the selective TRPV4 agonist GSK1016790A (Cayman Chemical) or DMSO as a vehicle and then incubated for 30 minutes at 37 °C. Following incubation, neutrophils were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) and blocked with 10% donkey serum in PBS overnight at 4 °C. Cells were stained with rabbit anti-H3-Cit antibody (Abcam No. 5103), washed, and incubated with anti-rabbit IgG AF488 (Invitrogen No. A21206). This was followed by washing and subsequent staining with AF647-conjugated myeloperoxidase (Abcam No. 252131). Coverslips were washed and mounted to slides using ProLong Gold Antifade Mountant with DAPI (Invitrogen). Images were collected with a Leica DM6000 microscope at 10× magnification with LasX software. Five fields of view were captured per slide, and NET quantitation was performed as area of colocalization of DAPI, H3-Cit, and MPO using Imaris Cell Imaging Software (Oxford Instruments). For the individual marker H3-Cit, captured images were analyzed in ImageJ (National Institutes of Health). Maxima count (prominence >10.00) for all images was normalized to the number of neutrophils plated.
5
Nanoscale Liposome Imaging via STED Microscopy
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All the samples were mounted in Abberior MOUNT SOLID Antifade medium (Abberior GmbH). STED images were acquired using a Leica SP8 STED 3× (Leica Microsystems) equipped with a pulsed white light laser as an excitation source and a 775 nm pulsed laser as depletion light source. All images were acquired at 400 Hz through a 100×/1.4 numerical aperture oil objective using the LAS X software (Leica Microsystems). mCherry-Sec61 was imaged in a confocal mode using an excitation wavelength at 561 nm. Liposomes doped with 0.5 mol% ATTO647N-PE were imaged using an excitation wavelength at 633 nm. The pixel resolution is 17 and 30 nm for dimensional and tridimensional STED images, respectively. All images were subjected to deconvolution using the Huygens Core, version 21.04/Huygens Remote Manager, version 3.7 (Scientific Volume Imaging; http://svi.nl/ ), using the classic maximum likelihood estimation algorithm, with 40 iterations to achieve a resolution with a signal-to-noise ratio equal to 20 for the confocal mode and five iterations to reach a resolution with a signal-to-noise ratio equal to 100 for the STED mode. Imaris Cell Imaging software (Oxford Instruments) was used to create tridimensional views from STED images and to measure liposome volume.
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