Using 96-well flat bottom plates (Nunc), canine PMN (n = 3, 2 × 105 in duplicates) were cocultured with D. immitis microfilariae (100 and 300 larvae per sample) or L3 (10 and 20 larvae per sample) in RPMI 1640 medium (1% penicillin/streptomycin, without phenol red) in a final volume of 200 µl. In parallel, PMN were pretreated with the NADPH inhibitor diphenyleneiodonium (DPI, 10 µM 30 min; Sigma-Aldrich) before exposure to microfilariae. To resolve NET formation, treatment with DNase I (90 U/sample, Roche Diagnostics) was used. For heat-induced killing (HI), microfilariae or L3 were incubated for 60 min at 60°C. Following coculture, 3 µM Sytox Orange® Nucleic Acid Stain (Life Technologies, final concentration) was added to each well, and measurements were performed every 30 min for up to 8 h. Sytox Orange-derived fluorescence intensities were estimated by spectrofluorometric analyses at an excitation wavelength of 547 nm and emission wavelength 570 nm using an automated plate monochrome reader (Varioskan Flash®; Thermo Scientific). For negative controls, PMN in plain medium were used. Stimulation of PMN with zymosan (1 mg/ml; Invitrogen) served as positive control.
Sytox orange nucleic acid stain
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
SYTOX Orange Nucleic Acid Stain is a fluorescent dye that binds to nucleic acids. It is designed for use in flow cytometry, fluorescence microscopy, and other applications that require the detection of DNA or RNA.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510, by Zeiss (4 mentions)
Reddot1 far red nuclear stain, by Biotium (4 mentions)
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Bz x710, by Keyence (3 mentions)
Anti dna histone 1 antibody, by Merck Group (2 mentions)
Mithras lb940 microplate reader, by Berthold Technologies (2 mentions)
Dnase 1, by Roche (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Calcium ionophore a23187, by Merck Group (2 mentions)
Ssrna40, by InvivoGen (2 mentions)
Prolong diamond antifade mountant with dapi, by Thermo Fisher Scientific (2 mentions)
Goat anti rat igg h l, by Abcam (2 mentions)
Donkey anti rabbit igg h l, by Abcam (2 mentions)
Donkey anti mouse igg h l, by Thermo Fisher Scientific (2 mentions)
Rabbit anti lyz, by Agilent Technologies (2 mentions)
Rabbit anti olfm4, by Cell Signaling Technology (2 mentions)
Recombinant rabbit anti aldolase b aldolase c, by Abcam (2 mentions)
Mouse anti human krt20, by Agilent Technologies (2 mentions)
Mouse anti chr a, by Santa Cruz Biotechnology (2 mentions)
34 protocols using sytox orange nucleic acid stain
1
Canine PMN Interactions with Dirofilaria Larvae
2
Osteoblast-Mediated Regulation of Bone Marrow Cell Apoptosis
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To test whether osteoblasts could affect the apoptosis of BMCs, osteoblasts from WT or ColI-Synd2 bones were plated in porous inserts (1 μm) and cultured to confluence. BMCs from WT mice were cultured on sterile coverslips in 24-well plates with or without osteoblasts in DMEM/FCS/P/S containing 15% L (control) or Wnt3a-CM for 72 h. The inserts were then removed and 0.5 μM SYTOX Orange nucleic acid stain (Life Technologies, Thermofisher, Waltham, MA, USA) was added to the medium. BMCs exposed to UV light were a positive control for apoptosis induction. The cells were washed and fixed with 4% paraformaldehyde in phosphate-buffered saline and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen). The ratio of Sytox- to DAPI-positive cells was measured under a fluorescent microscope with the Apotome 1 system (Zeiss) and Axiovision software.
3
Quantifying NET Formation via Immunostaining
4
Quantifying NET Formation via Immunostaining
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The immunostaining of extracellular chromatin fiber with anti-DNA/histone 1 antibody (Merck Millipore, Darmstadt, Germany, www.emdmillipore.com ) was used to demonstrate the NET formation. The fluorescence intensity of Sytox orange nucleic acid stain (Life Technologies) was used to quantify the NET measured with Mithras LB940 microplate reader (Bert-hold Technologies, Bad Wildbad, Germany, https://www.berthold.com/ ), at excitation and emission wavelengths of 540 nm and 580 nm, respectively. The details are presented in Supporting Information Experimental Procedures .
5
Visualizing NETosis in Neutrophils
6
Visualizing Root Tip Cell Death
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Cell death in the root tip was stained with 15 µM PI (Sigma) or 250 nM Sytox Orange nucleic acid Stain (Molecular Probes, Invitrogen) for 10 min, and rinsed twice with water before imaging with an LSM 700 confocal microscopy39 (link).
7
Visualizing NETosis in Neutrophils
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Purified genital neutrophils were re-suspended in HBSS containing Sytox orange nucleic acid stain (Molecular Probes, Eugene, OR) and plated on coverslips. HIV-VLP GFP-labeled (5,000 VLP/cell, unless otherwise specified), calcium ionophore A23187 (Sigma), or ssRNA40 (InvivoGen, San Diego, CA) were used to stimulate NETosis for 1h at 37 °C. Unstimulated neutrophils incubated in HBSS with Sytox orange were used as unstimulated controls. Cells were carefully washed with HBSS, and samples were mounted in Pro-Long Diamond anti-fade mountant with DAPI (Thermo Fisher Scientific). Samples were imaged using a Zeiss LSM 510 laser-scanning confocal microscope in combination with Zeiss Zen software.
8
Neutrophil NET Formation Assay
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Wells of 96-well black transparent-bottom plates (Greiner Bio-One, Kremsmünster, Austria) were either left untreated or coated overnight with 50 µg/ml FH in modified Hank's buffer.
Neutrophils (10 6 cells) were allowed to adhere to the wells for 30 min at 37°C in CO2 thermostat.
Soluble FH (50 µg/ml) or 100 nM PMA (Sigma-Aldrich) as a positive control was added and after 3 h of incubation in CO2 thermostat at 37°C, NETs were visualized on adherent neutrophils by addition of 5 µM Sytox Orange nucleic acid stain (Molecular Probes-Invitrogen).
Neutrophils (10 6 cells) were allowed to adhere to the wells for 30 min at 37°C in CO2 thermostat.
Soluble FH (50 µg/ml) or 100 nM PMA (Sigma-Aldrich) as a positive control was added and after 3 h of incubation in CO2 thermostat at 37°C, NETs were visualized on adherent neutrophils by addition of 5 µM Sytox Orange nucleic acid stain (Molecular Probes-Invitrogen).
9
Neutrophil Extracellular Traps Formation Assay
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Six treatments were prepared in a final volume of 150 µL as follows: T1 = PMN (control): 75 µL of PMN + 75 µL of RPMI-1640 medium [30 (link)]; T2 = PMN + Whole semen (sperm + SP): 75 µL of PMN + 75 µL of Whole semen [10 (link)]; T3 = PMN + Sperm: 75 µL of PMN + 75 µL of Sperm [30 (link)]; T4 = PMN + SP: 75 µL of PMN + 37.5 µL of SP (25% of SP with respect to the total volume) + 37.5 µL of RPMI-1640 medium [30 (link)]; T5 = PMN + formyl-methionyl-leucyl-phenylalanine (FMLP): 75 µL of PMN + 15 µL 0.1 mM FMLP (F3506; Sigma-Aldrich, St. Louis, MO, USA) + 60 µL of RPMI-1640 medium [10 (link),21 (link),30 (link)]; and T6 = PMN + Kenney: 75 µL of PMN + 75 µL of Kenney [30 (link)].
All treatments were incubated at 37 °C for 2 h. After that, PMN were stained with SYTOX™ Orange Nucleic Acid Stain (S11368; ThermoFisher Scientific, Waltham, MA, USA) and the percentage of reacted PMN (NETosis) was evaluated using a confocal microscope. Extracellular ROS production (measured as H2O2 levels) was determined through the Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit (A22188; ThermoFisher Scientific, Waltham, MA, USA). The evaluations were carried out by the same technician to avoid errors and biases due to the human factor.
All treatments were incubated at 37 °C for 2 h. After that, PMN were stained with SYTOX™ Orange Nucleic Acid Stain (S11368; ThermoFisher Scientific, Waltham, MA, USA) and the percentage of reacted PMN (NETosis) was evaluated using a confocal microscope. Extracellular ROS production (measured as H2O2 levels) was determined through the Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit (A22188; ThermoFisher Scientific, Waltham, MA, USA). The evaluations were carried out by the same technician to avoid errors and biases due to the human factor.
10
Multicolor Immunostaining Protocol
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The primary antibodies used in this study are as follows: rabbit anti-Lyz (1:800; Dako, #A0099), rabbit anti-Olfm4 (1:500; Cell Signaling Technology, #39141), recombinant rabbit anti-aldolase B + aldolase C (1:300; Abcam, #ab75751), mouse anti-human KRT20 (1:500; Dako, #M701929-2), mouse anti–Chr-A (1:50; Santa Cruz Biotechnology, #sc-393941), rat anti–E-cadherin (1:400; Santa Cruz Biotechnology, #sc-59778), CD24 Monoclonal Antibody (1:200; Thermo Fisher Scientific, #17-0242-80), and mouse anti-Ki67 (1:200; BD Biosciences, 550609). The secondary antibodies used in this study are as follows: Goat Anti-Rabbit IgG H&L (Alexa Fluor 405) preadsorbed (1:1000; Abcam, #ab175654), Goat Anti-Rat IgG H&L (Alexa Fluor 555) preadsorbed (1:1000; Abcam, #ab150166), Donkey Anti-Mouse IgG H&L (Alexa Fluor 647) (1:500; Thermo Fisher Scientific, #A31571), and Donkey Anti-Rabbit IgG H&L (Alexa Fluor 405) preadsorbed (1:1000; Abcam, #ab175649). The dyes used in this study are as follows: WGA conjugated to CF488A (5 μg/ml; Biotium), RedDot1 Far-Red Nuclear stain (1:200; Biotium), and SYTOX Orange Nucleic Acid Stain (1:5000; Thermo Fisher Scientific, #S11368). The order of staining, optimized to ensure good staining quality for all cell types, was based on the staining quality and stripping difficulty of each antibody (fig. S1G).
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