Cells were fixed in 4% paraformaldehyde solution (PFA; Sigma-Aldrich, St. Louis, MO, USA) in phosphate-buffered saline (Thermo Fisher Scientific), permeabilized with PBS/0.1% Triton X-100 (Sigma-Aldrich) and blocked in 4% bovine serum albumin (BSA, Sigma-Aldrich) in PBS. Then, cells were incubated with the primary antibodies diluted in PBS with 4% BSA. Primary antibodies specific for Flavivirus envelope protein E (clone 4G2, mouse, 1:500, Merck Millipore, USA), PAX6 (rabbit, 1:100, Sigma-Aldrich), Nestin (mouse, 1:100, Abcam, Cambridge, UK) were used. Cells were incubated overnight at 4 °C. The secondary antibodies used included the anti-mouse IgG Alexa Fluor-488 (goat, 1:250, Thermo Fisher Scientific) and the anti-rabbit IgG Alexa Fluor-546 (goat, 1:250, Thermo Fisher Scientific). DRAQ5 fluorescent probe solution (Thermo Fisher Scientific) was used to stain nuclei. Immunofluorescence was visualized by a confocal microscope (Leica, Wetzlar, Germany) under 63× magnification.
Draq5 fluorescent probe solution
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
DRAQ5™ Fluorescent Probe Solution is a cell-permeable, far-red fluorescent DNA stain. It binds to the minor groove of double-stranded DNA, allowing for the visualization and quantification of DNA content in cells.
Automatically generated - may contain errors
Lab products found in correlation
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (3 mentions)
Paraformaldehyde solution, by Merck Group (2 mentions)
Nestin, by Abcam (2 mentions)
Confocal microscope, by Leica (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Steve software, by Nanolive (2 mentions)
Er ccd camera, by Nikon (2 mentions)
Ti e inverted microscope, by Nikon (2 mentions)
Alexa fluor 488 anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 nhs ester, by Thermo Fisher Scientific (1 mentions)
Cho k1 cells, by American Type Culture Collection (1 mentions)
Cytochalasin d, by Thermo Fisher Scientific (1 mentions)
Wheat germ agglutinin alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Pierce ldh cytotoxicity assay kit, by Thermo Fisher Scientific (1 mentions)
F 12k medium, by American Type Culture Collection (1 mentions)
Glutamax supplement, by Thermo Fisher Scientific (1 mentions)
3d cell explorer fluo, by Nanolive (1 mentions)
Ma900, by Sony (1 mentions)
Zombie aqua fixable viability kit, by BioLegend (1 mentions)
15 protocols using draq5 fluorescent probe solution
1
Immunofluorescence Staining of Flavivirus Proteins
2
Inflammasome Activation Assay in THP-1 Cells
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The following reagents were purchased: phorbol 12-myristate 13-acetate (PMA) and CP-456773 sodium salt (CRID3) from Sigma-Aldrich (USA); Amicon Ultra 0.5 mL centrifugal filter MWCO 100 kDa, MF-Millipore membrane filters of 0.45 μm and 0.22 μm pore sizes from Millipore Sigma (USA); GM-CSF recombinant human protein, fetal bovine serum (FBS), penicillin-streptomycin, sodium pyruvate (100 mM), GlutaMAX supplement, RPMI 1640 medium, PBS buffer, Pierce LDH cytotoxicity assay kit, cytochalasin D (CytD), Alexa Fluor 488 NHS ester (succinimidyl ester), wheat germ agglutinin Alexa Fluor 555, DRAQ5 fluorescent probe solution (5 mM) and 4% formaldehyde from ThermoFisher Scientific (USA); acridine orange (AO) from Dojindo Laboratories (Kumamoto, Japan); VX765 and lipopolysaccharide (LPS) Escherichia coli O111:B4 from InvivoGen (USA); human IL1 beta assay kit from Cisbio Bioassays (France); CD14 microbeads (human) from Miltenyi Biotech (Germany); silica crystals (MIN-U-SIL-15) from US Silica (USA); μ-Slide 8 well ibiTreat from Martinsried (Germany); THP-1 cells from Japanese Collection of Research Bioresource Cell Bank (Japan); CHO-K1 cells and F-12K medium from ATCC (USA).
Human PBMCs were donated from University Hospital Bonn (Germany), and the operation and usage were approved by the local ethics committee in accordance with the Declaration of Helsinki.
Human PBMCs were donated from University Hospital Bonn (Germany), and the operation and usage were approved by the local ethics committee in accordance with the Declaration of Helsinki.
3
Holotomographic Imaging of Cells
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Holotomographic images were obtained by using 3D Cell Explorer-fluo (Nanolive) microscope equipped with an 60x magnification (λ = 520 nm, sample exposure 0.2 mW/mm2) and a depth of field of 30 µm. For host cell nuclei visualization, BUVEC were stained with the vital dye DRAQ5 (DRAQ5™ Fluorescent Probe Solution, ThermoFischer). Images were analyzed using STEVE® software (Nanolive) to obtain a refractive index-based z-stack56 and digital staining was applied according to the refractive index of intracellular structures.
4
Isolation of Mouse Brain Microglia
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Mice were euthanized with Euthanyl Reg and transcardially perfused with ice-cold HBSS (Invitrogen 14175103). Brains were dissected, processed, and diced in ice-cold HBSS. Collected tissue was then dounced 5 times with a loose fitted dounce and subsequently filtered through 70 and 20 μm cell strainers. Cells were pelleted and myelin debris was removed with Debris Removal Solution (Miltenyi Biotech 130–109-398). Cells were then blocked for 5 minutes with Anti-Mo CD16/CD32 (Invitrogen 14–0161-85) and stained using Zombie Aqua Fixable Viability Kit (Biolegend 423102), DRAQ5 Fluorescent Probe Solution (Thermofisher 62254) and FITC Mouse anti Rat CD11b (BD Biosciences 561684) for 20 min. After cell staining, cells were sorted on a Sony MA900. Live nucleated single cells that were CD11b positive were gated and collected. Final collected cell counts were determined using a hemocytometer.
5
Sperm Cell Fixation and Analysis
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We fixed the sperm following the published protocol83 by adding 16 μl of 50 mM EDTA pH 8 per milliliter of cell suspension (48 μl for 3 ml) so as to obtain a final concentration of 0.8 mM EDTA, and then to fix germ cells slowly adding 3 volumes of ice-cold 100% ethanol using vortex at low speed. The cells are stained with Draq5 DNA stain (DRAQ5™ Fluorescent Probe Solution, Cat# 62251, ThermoFisher Scientific, used at 50 nM final concentration) and data was collected using ImageStream (Luminex Corporation, Austin, TX). Each event was simultaneously imaged in bright field for morphology, in Draq5 fluorescent channel to measure DNA content, and in dark field channel to measure side scatter. By plotting total Draq5 intensity, to differentiate 1n sperm cells and 2n somatic cells, versus Draq5 channel Modulation feature measurement (that measures the intensity range of an image, normalized between 0 and 1) we were able to identify six general population which contained: small debris, cell fragments and larger debris, sperm cells, sperm cells aggregates, somatic cells, and larger aggregates. The analysis was done using IDEAS 6.2 software (Luminex Corporation, Austin, TX).
6
Immunohistochemical and Immunofluorescent Analysis
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Tissue sections of 5 µm thickness were prepared and stained with Hematoxylin–Eosin (HE). The alternative sections were used for immunohistochemical and immunofluorescent analysis. The following primary antibodies were applied for the detection of specific protein expression by immunohistochemistry (Table 1 ) or immunofluorescence (Table 2 ): β-Catenin PY 489, FZD6, pan-cytokeratin, Sex determining region Y box 2 (SOX2), Ki-67. In the case of immunohistochemistry, 3,3′-Diaminobenzidine (DAB) was used for visualization of the signal and Hematoxylin was used to counterstain the nuclei. For visualization of nuclei in the case of immunofluorescence, DRAQ5™ Fluorescent Probe Solution was used (Thermo Scientific™, Shanghai, China). Sections were photographed under bright-field illumination with a Leica compound microscope DMLB2 (Leica, Wetzlar, Germany) or SP8 Resonant Scanning Confocal microscope (Leica, Wetzlar, Germany) in the case of immunofluorescence.
7
Mitochondrial Function Assay Protocol
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Cells were costained with 100 nM MitoTrackerTM CMTMRos Orange (Invitrogen) and DRAQ5 Fluorescent Probe Solution (62251) (Thermo ScientificTM) for 30 min. Mitochondria images, number, and intensity were acquired by PerkinElmer Operetta and analyzed by harmony analysis software. Loss functional mitochondria was monitored by the sensitivity to carbonilcyanide p-triflouromethoxyphenylhydrazone (FCCP).
8
Holotomographic Imaging of Cellular Structures
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3D-holotomographic images were obtained by using 3D Cell Explorer microscope (Nanolive® 3D) equipped with an × 60 magnification optic (λ = 520 nm, sample exposure 0.2 mW/mm2) and a field depth of 30 μm. Images were analyzed using the STEVE® software (Nanolive) to obtain a refractive index-based z-stack. Digital staining was applied according to the refractive index of intracellular structures. Nuclei were stained by DRAQ5 probe for vital DNA staining following the manufacturer instructions (DRAQ5™ Fluorescent Probe Solution, ThermoFischer).
9
Hippocampal Amyloid-Beta Immunohistochemistry
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Paraffin-embedded blocks were prepared by sequential dehydration in graded ethanol and infiltration in paraffin before embedding. Blocks were serially sectioned between –1.2 mm and –2.4 mm from Bregma at a thickness of 5 μm. Dewaxed and rehydrated hippocampal sections were first boiled in Tris-EDTA buffer, pH 9.0, and incubated with 0.1 M glycine in PBS for 20 min to reduce autofluorescence. Tissues processed for Aβ staining were pretreated with 99% formic acid for 7 min. Sections were then incubated overnight at 4°C with the antibodies listed in Table 4 . After washing in PBS, the immunoreaction was visualized using Alexa Fluor 488, 555, and 594 conjugated secondary antibodies (1:400; Thermo Fisher Sci., A27012, A27017, A11005, respectively). Nuclei were stained with DRAQ5 fluorescent probe solution (Thermo Fisher Sci., 62251). Neurons were fixed for 15 min with 4% paraformaldehyde (Thermo Fisher Sci., 28908) and permeabilized with 0.1% saponin (Sigma-Aldrich, 47036) in blocking buffer (1% BSA + 20 mM glycine in PBS) for 15 min before proceeding with the immunostaining. Confocal images were collected with a Leica TCS SPE confocal laser scanning microscope (Leica Microsystems, Barcelona, Spain) using a 63x/1.32-0.60 oil PH3 CS objective and a confocal pinhole set at 1 Airy unit. All confocal images shown are single optical sections.
10
Multicolor Imaging of Subcellular Structures
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U2OS-LMNB1-TUBA1B-ACTB cells were incubated with an ER staining solution (1:1000 v/v dilution of stock solution, ER-Tracker™ Blue-White DPX, Thermo Fisher Scientific) in cell culture medium (including 2% FBS) for 35 min under standard cell culture conditions. Next, the staining solution was removed and cells were incubated with 25 µl of paraformaldehyde solution per well (4%; Sigma Aldrich) for 20 min at room temperature. After a washing step with 60 µl of PBS, cells were stained using 16 µl of the nuclear staining reagent DRAQ5 (2.5 µM, DRAQ5™ Fluorescent Probe Solution, Thermo Fisher Scientific) per well for 60 min. After removal of the staining solution 60 µl of PBS were added per well, plates were sealed and kept in a fridge until imaging.
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