The cytosolic Ca2+ levels and the ROS production were measured in living SH‐SY5Y cells plated in 12‐well plates containing coverslips at the density of 150 000 cells/well. After 24 h the cells were washed with PBS and incubated with no treatment with 1 μM ionomycin for 1 h for the Ca2+ level detection or 250 μM H2O2 for 1 h for the ROS production assay, and with 5 μM trodusquemine for different times. Cells were then loaded with 4 μM Fluo‐4 AM (Thermo Fisher Scientific) for 10 min after the treatments, to detect the Ca2+ ions, or with 5 μM CM‐H2DCFDA (Thermo Fisher Scientific) the last 15 min of treatments, to detect ROS. Cells were analyzed using a TCS SP8 scanning confocal microscopy system equipped with an argon laser source (Leica Microsystems, Mannheim, Germany), after excitation at 488 nm. A series of 1‐μm‐thick optical sections (1024 × 1024 pixels) was taken using a Leica Plan Apo 63× oil immersion objective and projected along the z‐axis as a single composite image. >10 cells, in three different experiments, were analyzed using ImageJ software.
Plan apo 63
Manufactured by Leica
Sourced in Germany
The Plan Apo 63x is a high-quality objective lens designed for microscopy applications. It features a plan-apochromatic optical design, which provides excellent flatness of field, color correction, and resolution. This objective is suitable for a variety of microscopy techniques and can be used with various sample types.
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Lab products found in correlation
Tcs sp8 scanning confocal microscopy system, by Leica (3 mentions)
Cm h2dcfda, by Thermo Fisher Scientific (2 mentions)
Fluo 4 am, by Thermo Fisher Scientific (2 mentions)
Lsm 800 airyscan, by Zeiss (1 mentions)
Fam devd fmk, by ImmunoChemistry Technologies (1 mentions)
Tcs sp8, by Leica (1 mentions)
Bodipy 581 591 c11, by Thermo Fisher Scientific (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Tcs sp5 clsm, by Leica (1 mentions)
Lysotracker red dnd 99, by Thermo Fisher Scientific (1 mentions)
Tcs sp5 scanning microscope, by Leica (1 mentions)
Fluo 3 am, by Thermo Fisher Scientific (1 mentions)
Mitosox red mitochondrial superoxide indicator, by Thermo Fisher Scientific (1 mentions)
Eclipse ti, by Yokogawa (1 mentions)
γh2ax, by Merck Group (1 mentions)
Lasx 3, by Leica (1 mentions)
Lasaf 2, by Leica (1 mentions)
Tcs sp5 2 confocal, by Leica (1 mentions)
Formalin, by Merck Group (1 mentions)
Alexa488 conjugated donkey anti goat, by Thermo Fisher Scientific (1 mentions)
14 protocols using plan apo 63
1
Cytosolic Ca2+ and ROS Levels in SH-SY5Y Cells
2
Confocal Microscopy Imaging of Fluorescent Cells
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Images of immunostained cells or cells expressing fluorescent-tagged proteins were acquired on Confocal inverted microscope SP8-X (DMI 6000; Leica). Optical sections were acquired with a Plan Apo 63× oil immersion objective (N.A. 1.4; Leica) using the LAS-X software. Fluorescence was excited using either a 405-nm laser diode or a white light laser and later collected after adjusting the spectral windows with GaAsP PMTs or Hybrid detectors. Images from a mid-focal plane are shown. For Huh7 experiments, images were acquired by the ZEISS LSM800 Airyscan.
3
HEWL Aggregates Induce Caspase-3 Activity
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HEWL aggregates were formed in the absence or in the presence of 0.32 µM of compounds 1 –6 as described in the previous section and then added to the cell culture medium of SH-SY5Y cells seeded on glass coverslips for 24 h at 2 μM (monomer equivalents). Caspase-3 activity was then analyzed by using FAM FLICATM caspases 3 & 7 solution (caspase 3 & 7 FLICA kit FAM-DEVDFMK, Immunochemistry Technologies, LLC, Bloomington, MN, USA). Cell fluorescence was analyzed by the TCS SP8 scanning confocal microscopy system (Leica Microsystems, Mannheim, Germany) equipped with an argon laser source, as previously reported [62 (link)]. A series of 1.0 μm thick optical sections (1024 × 1024 pixels) was taken through the cell depth for each sample using a Leica Plan Apo 63 × oil immersion objective for fluorescence measurement at 488 nm. The confocal microscope was set at optimal acquisition conditions, e.g., pinhole diameters, detector gain, and laser powers. Settings were maintained constant for each analysis.
4
Confocal Lipid Peroxidation Quantification
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Lipid peroxidation, a reliable oxidative stress index, was assayed by confocal scanning microscopy using BODIPY 581/591 C11 (Life Technologies, Carlsbad, CA, USA), a lipophilic fluorescent probe that mimics the properties of natural lipids [13 (link)]. In the presence of oxidizing agents, BODIPY 581/591 C11 shifts its fluorescence from red to green. Cells, cultured on glass coverslips, were loaded with BODIPY dissolved in 0.1% DMSO (2.5 μmol L−1 final concentration) for 15 min at 37 °C in DMEM. Cells were then fixed in 2.0% buffered paraformaldehyde for 10 min at room temperature and fluorescence estimated using a confocal Leica TCS SP8 scanning microscope equipped with an argon laser source for fluorescence measurements. A series of optical sections (1024 × 1024 pixels) 1.0 μm in thickness was taken throughout the cell depth at intervals of 0.5 μm using a Leica Plan Apo 63× oil immersion objective and then projected as a single composite image by superimposition. Lipid peroxidation was also quantified by flow cytometry on single-cell suspensions incubated (30 min, 37 °C in the dark) with BODIPY 581/591 (2.5 μmol L−1) in DMEM, washed, resuspended in PBS and analyzed under a FACSCantoII flow cytometer (Becton Dickinson, San Jose, CA, USA).
5
Quantifying Phagolysosome Fusion in Macrophages
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J774 macrophages were seeded onto CLSM culture dishes (Mat Tech, United States) at a concentration of 2.5 × 105 cells per dish. Those macrophages were cultured in DMEM with LysoTracker Red DND-99 (Life Technologies, United States) at a concentration of 1:10000. The 24 h after the seeding, cells were placed in contact with beads in order to compare the phagolysosome fusion between macrophages with uncoated beads versus macrophages with TPP-coated beads. After 24 h, dishes were observed using a TCS-SP5 CLSM (Leica, Germany) with a PlanApo 63 (NA, 1.4) oil objective, operating at a zoom of 2.5. One hundred macrophages were counted for each sample, and colocalization between LysoTracker and the green fluorescence produced by the beads was calculated using Pearson’s correlation coefficient.
6
Visualizing α-Synuclein Aggregation in SH-SY5Y Cells
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SH-SY5Y cells seeded on glass coverslips were incubated for 60 min with αS conformers (0.3 μM) and then stained with 5.0 μg/mL Alexa Fluor 633-conjugated wheat germ agglutinin (Thermo Fisher Scientific), as previously reported [20 (link)]. Following a fixing step with 2% (v/v) paraformaldehyde, and a permeabilization phase with glycerol at 3.0% (v/v), αS was detected with rabbit polyclonal anti-oligomer A11 antibodies (1:250), rabbit polyclonal anti-amyloid fibrils OC (1:800), or with mouse monoclonal anti-αS 211 antibodies (1:250), then with Alexa-Fluor-488-conjugated anti-rabbit or mouse secondary antibodies (1:1000, Thermo Fisher Scientific). The detection of the fluorescence emission was obtained by double excitation at 633 nm and 488 nm through a TCS SP8 scanning confocal microscopy system (Leica Microsystems, Mannheim, Germany). A series of 1.0 μm thick optical sections (1024 × 1024 pixels) was taken through the cell depth for each sample using a Leica Plan Apo 63 × oil immersion objective and projected as a single composite image by superimposition. The confocal microscope was set and maintained constant at optimal detector gain and laser powers.
7
Calcium Imaging of H3 Peptide Effects
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Aggregates of the H3 wt, H3 mutA and H3 mutE peptides (1 mM monomer concentration) were incubated in 50 mM sodium phosphate buffer, pH 7.2, 25°C under stirring, for 0 h, 15 days and 30 days. The samples were then diluted in cell culture media at a 100 μM peptide concentration, and then added to the SH-SY5Y cells seeded on glass coverslips for 60 min at 37°C. The cells were then loaded for 30 min at 37°C with 10 μM fluo3-AM (Life technologies, CA, USA), as previously described [27 ]. The resulting cell fluorescence was analysed by confocal Leica TCS SP5 scanning microscope (Mannheim, Germany) equipped with an argon laser source for fluorescence measurements at 488 nm and a Leica Plan Apo 63× oil immersion objective. A series of optical sections (1024 × 1024 pixels) 1.0 μm in thickness was taken through the cell depth for each examined sample. The confocal microscope was set at optimal acquisition conditions, e.g. pinhole diameters, detector gain and laser powers. Settings were maintained constant for each analysis. To quantify the signal intensity of the fluorescent probe between 10 and 22 cells were analysed using ImageJ software (NIH, Bethesda, MD) and the fluorescence intensities expressed as arbitrary units.
8
Mitochondrial Superoxide Detection in NSC-34 Cells
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Mitochondrial superoxide ion production was detected with a static time point measurement in living NSC-34 cells by using the MitoSOX Red Mitochondrial Superoxide Indicator (TM36008, Thermo Fisher Scientific). Cells were plated in 12-well plates containing coverslips and transiently transfected as described above (see the “Transient transfection” section). At different lengths of time after transfection (0 to 72 hours), cells were treated with the MitoSOX probe for 10 min. Fluorescence emission was detected after excitation at 550 nm by the TCS SP8 scanning confocal microscopy system (Leica Microsystems) equipped with an argon laser source. A series of 1.0-μm-thick optical sections (1024 × 1024 pixels) was taken through the cell depth for each sample using a Leica Plan Apo 63× oil immersion objective and projected as a single composite image by superimposition. The confocal microscope was set at optimal acquisition conditions, e.g., pinhole diameters, detector gain, and laser powers. Settings were maintained constant for each analysis.
9
Spatiotemporal Dynamics of MRE11 Mutants
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U2OS cells were transiently transfected with pEYFP-MRE11 (WT), MRE11S676AS678A (MUT), MRE11S676DS678D (DD), MRE11S676A (S676A) or MRE11S678A (S678A) plasmids respectively using electroporation (Amaxa Nucleofector, Germany) 24 h prior to irradiation. Charged particle irradiation was done at the UNILAC accelerator at GSI using 9.8 MeV/u carbon ions (LET 170 keV/μm) or 4.7 MeV/u uranium ions (LET 15000 keV/μm) under a low angle as described previously (43 (link)). Cells were fixed with 2% paraformaldehyde unirradiated or 10 min, 1, 4 or 12 h post-irradiation. Immunostaining with γH2AX (Millipore) was done according to Jakob et al. (44 (link)). DNA was counterstained with 1 μg per ml DAPI. Microscopic imaging was performed using a spinning disc confocal microscope (Nikon Eclipse Ti with Yokogawa CSU_X1) utilizing a Plan APO 100 × 1.4 NA oil immersion lens or a Leica SPE laser scanning confocal (Planapo 63× 1.3 NA). Optical sections were recorded in increments of 300 μm across the thickness of cells. The total magnification of the systems yielded pixels corresponding to 72 × 72 nm in lateral dimensions. Real time recruitment kinetics of wild-type (WT) and mutant YFP-MRE11 after carbon or uranium ion irradiation was evaluated using the GSI beamline microscope as described previously (45 (link)). About 32 to 55 nuclei were analysed for each plasmid (8 samples for each condition).
10
Live Cell Imaging Microscopy Protocol
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Live cell imaging was performed using a Leica TCS SP5 II confocal laser scanning microscope with a Plan Apo 63 × 1.4 NA oil-immersion objective and an environmental cell culture chamber as in our previous work17 (link),42 .
The fluorescence imaging was taken using excitation/emission (ex/em) wavelengths as follows:
The fluorescence imaging was taken using excitation/emission (ex/em) wavelengths as follows:
Atto520 channel, ex/em range = 514/520–640 nm.
tC/Atto520 channel, ex/em range = 405/520–640 nm.
FAM channel, ex/em range = 480/510–540 nm.
TAMRA channel, ex/em range = 560/595–630 nm.
FRET channel, ex/em range = 480/595–630 nm.
Fluorescence channel for DNA-AgNCs, ex/em range = 500/510–550 nm.
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