We assessed human NET formation as previously described.18 (link) We treated PMNs isolated from healthy adults and COVID-19 patients with phorbol 12-myristate 13-acetate (PMA; 20 nM; 2 hours), polyinosinic-polycytidylic acid [poly(I:C); 1 μM; 2 hours], or COVID-19 patient plasma (undiluted; 2 hours), with and without a 1-hour pretreatment with nNIF or its inactive scrambled peptide control (1 nM). For all experiments, we visualized NET formation using confocal microscopy (Olympus) with cell-permeable (SYTO Green) or cell-impermeable (SYTOX Orange; both from Molecular Probes) DNA fluorescent dyes. We quantitatively assessed NET formation using a cell-free DNA fluorescence assay with SYTOX Green DNA dye. Relative fluorescence was quantified using a fluorometric plate reader with SoftMax software (Molecular Devices).21 (link) Given the challenges of working in a Biosafety Level 2 Plus facility, our in vitro studies of NET formation relied on the combination of confocal imaging with representative images and a high-throughput cell-free DNA fluorescence assay quantitation.
Syto green
Manufactured by Thermo Fisher Scientific
SYTO Green is a nucleic acid stain used for fluorescent labeling and visualization of DNA and RNA in various biological samples. It has excitation and emission wavelengths suitable for detection with common fluorescence microscopy and flow cytometry instrumentation.
Automatically generated - may contain errors
Lab products found in correlation
Sytox orange, by Thermo Fisher Scientific (9 mentions)
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Micrococcal dnase, by Worthington (3 mentions)
Poly l lysine (pll), by Merck Group (3 mentions)
Sytox green, by Thermo Fisher Scientific (3 mentions)
Sivelestat, by Merck Group (2 mentions)
Medium 199, by Lonza (2 mentions)
Meo sucaapv pna, by Merck Group (2 mentions)
Cytochalasin b, by Merck Group (2 mentions)
Anti cd15 microbeads, by Miltenyi Biotec (2 mentions)
Cytochalasin d, by Merck Group (2 mentions)
Paraformaldehyde pfa, by Merck Group (2 mentions)
Fv1000 ix81 confocal microscope, by Olympus (2 mentions)
Fluoview software, by Olympus (2 mentions)
Sybr green 2, by Thermo Fisher Scientific (2 mentions)
Thioflavin t, by Merck Group (2 mentions)
Softmax software, by Molecular Devices (1 mentions)
Thrombin, by Merck Group (1 mentions)
Dnase, by Promega (1 mentions)
Lipopolysaccharide, by Merck Group (1 mentions)
16 protocols using syto green
1
Quantifying NET Formation in COVID-19
2
Neutrophil Activation and Extracellular Traps
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Example 8
Lipopolysaccharide (E. coli serotype 0111:134 and Salmonella enteritidis), poly-L-lysine, cytochalasin B, cytochalasin D, paraformaldehyde (p-FA), sivelestat, NE, the NE substrate (MeOSuc)-AAPV-(pNA), and thrombin were purchased from SIGMA-ALDRICH®. Additional reagents were: TO-PRO®-3 stain, phalloidin, SYTO® Green (cell permeable DNA stain), and SYTOX® Orange (cell impermeable DNA stain) (MOLECULAR PROBES®); Cl-amidine (CALBIOCHEM®); DNase (PROMEGA™); Anti-CD15-microbeads (MILTENYI™); Medium-199 (LONZA™) and micrococcal DNase (WORTHINGTON®).
3
Visualizing Neutrophil Extracellular Traps
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Example 11
Qualitative assessment of NET formation was performed as previously reported (see Yost C C, et al., Blood. 2009; 113 (25):6419-27 and McInturff A M, et al., Blood. 2012; 120 (15):3118-25). Briefly, primary PMNs isolated from preterm infants, healthy term infants, and healthy adults (2×106 cells/mL) were incubated with control buffer or stimulated with indicated agonists or bacteria for 1 hour at 37° C. in 5% CO2/95% air on glass coverslips coated with poly-L-lysine. For selected experiments, primary PMNs were pre-incubated with autologous plasma, cord blood plasma, nNIF (0.2-70 nM), CRISPP (0.2-70 nM), nNIF-SCR (1 nM), or CRISPP-SCR (1 nM) for one hour prior to stimulation. After pre-incubation and/or stimulation, PMNs were gently washed with PBS and incubated with a mixture of cell permeable (SYTO® Green, MOLECULAR PROBES®) and impermeable (SYTOX® Orange, MOLECULAR PROBES®) DNA fluorescent dyes. Confocal microscopy was accomplished using a FV1000 IX81 confocal microscope and FLUOVIEW™ software (OLYMPUS™). Both 20× and 60× objectives were used. Z-series images were obtained at a step size of 1 μm over a range of 20 μm for each field. FLUOVIEW™ and ADOBE™ PHOTOSHOP™ CS2 software was used for image processing.
4
Immunological Assays with LPS and Cytoskeletal Modulators
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Example 8
Lipopolysaccharide (E. coli serotype 0111:134 and Salmonella enteritidis), poly-L-lysine, cytochalasin B, cytochalasin D, paraformaldehyde (p-FA), sivelestat, NE, the NE substrate (MeOSuc)-AAPV-(pNA), and thrombin were purchased from SIGMA-ALDRICH® Additional reagents were TO-PRO®-3 stain, phalloidin, SYTO® Green (cell permeable DNA stain), and SYTOX® Orange (cell impermeable DNA stain) (MOLECULAR PROBES®). Cl-amidine (CALBIOCHEM®). DNase (PROMEGA™); Anti-CD15-microbeads (MILTENYI™); Medium-199 (LONZA™), and micrococcal DNase (WORTHINGTON®).
5
Quantitative Analysis of NET Formation
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Qualitative assessment of NET formation was performed, as previously referenced (10 (link), 17 (link)). Briefly, participant primary PMNs were incubated with control buffer or stimulated with LPS (100 ng/mL) for 2 h at 37°C in 5% CO2/95% air on glass coverslips coated with poly-l -lysine. After stimulation, PMNs were gently washed with PBS and incubated with a mixture of cell permeable (Syto Green, Molecular Probes) and impermeable (Sytox Orange, Molecular Probes) DNA fluorescent dyes. Confocal microscopy was accomplished using a FV300 1X81 Microscope and the FluoView software (Olympus). Both 20× and 60× objectives were used. Z-series images were obtained at a step size of 1 μm over a range of 20 μm for each field. The Olympus FluoView software and the Adobe Photoshop CS2 software were used for image processing. Semiquantitative analysis of NET formation was accomplished using the ImageJ analysis software (NIH) and a standardized grid system with rigorous NET quantitation. Statistical comparisons were made via one way ANOVA with Tukey’s multiple comparisons post hoc testing.
6
LPS-Induced Cellular Stress Assay
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We purchased lipopolysaccharide from Salmonella enterica serotype enteritidis (Sigma: L6011), poly-L-lysine (Sigma), SYTO Green (cell-permeable DNA stain; Molecular Probes), SYTOX Orange (cell-impermeable DNA stain; Molecular Probes), DNase 1 (Promega), micrococcal DNase (Worthington), isoflurane anesthesia from VETONE (FLURISO™), meropenem (Sargent®) from University of Utah Hospital, DMSO (Fisher Scientific: BP 231-1), and sterile isotonic saline (BD PosiFlush™;10 mL syringes).
7
Confocal Imaging of Neuroblastoma Cells
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Confocal images of human neuroblastoma (SH-SY5Y) cell cultures were captured in complete medium at 37 °C, using a laser scanning confocal microscope (Leica TCS SP2 AOBS equipped with a HCX PL APO 63 × 1.4 oil, immersion objective, Germany). Briefly, SH-SY5Y cells were seeded in 35 cm2 plates (Mat Tek) with approximately 30 % of confluence in complete medium and incubated for 72 h in the presence of sterile PBS buffer + PK (positive control) and the PK-resistant core of the PrPs IBs. Proteinase K was inactivated by boiling all solutions before applying them to cultured cells. Cells were incubated with 0.5 μg/mL SYTO green and 10 μg/mL propidium iodide (PI) (Molecular Probes) for 15 min at 37 °C and washed twice with PBS buffer. Cell morphology was analysed by confocal fluorescence microscopy using an orange diode (588–715 nm emission collected) and a UV laser (excited at 350 nm and collected at 405 nm). Two independent experiments, both in duplicate were done and the entire field of each plate was observed at the microscope.
8
Visualizing NET Formation in Infant PMNs
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Example 11
Qualitative assessment of NET formation was performed as previously reported (see Yost C C, et al., Blood. 2009; 113 (25) 6419-27 and McInturff A M, et al., Blood. 2012; 120 (15) 3118-25). Briefly, primary PMNs isolated from preterm infants, healthy term infants, and healthy adults (2×106 cells/mL) were incubated with control buffer or stimulated with indicated agonists or bacteria for 1 hour at 37° C. in 5% CO2/95% air on glass coverslips coated with poly-L-lysine. For selected experiments, primary PMNs were pre-incubated with autologous plasma, cord blood plasma, nNIF (0.2-70 nM). CRISPP (0.2-70 nM), nNIF-SCR (1 nM), or CRISPP-SCR (1 nM) for one hour prior to stimulation. After pre-incubation and/or stimulation, PMNs were gently washed with PBS and incubated with a mixture of cell permeable (SYTO® Green, MOLECULAR PROBES®) and impermeable (SYTOX® Orange, MOLECULAR PROBES®) DNA fluorescent dyes. Confocal microscopy was accomplished using a FV1000 IX81 confocal microscope and FLUOVIEW™ software (OLYMPUS™) Both 20× and 60× objectives were used Z-series images were obtained at a step size of 1 μm over a range of 20 μm for each field. FLUOVIEW™ and ADOBE™ PHOTOSHOP™ CS2 software was used for image processing.
9
Visualizing Peritoneal NET Formation
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We evaluated 7 to 10-day-old mice for peritoneal NET formation via live-cell imaging with confocal microscopy as previously described.22 (link) Sham and cecal slurry injected mice ± experimental conditions were euthanized 24 h after cecal slurry injection and peritoneal fluid samples were collected as described above. On a glass coverslip coated with poly-l -lysine, 100 μL of peritoneal fluid was incubated for 1 h at 37 °C in 5% CO2/95% air. DNA fluorescent stains for cell-permeable (SYTO Green, Molecular Probes) and cell-impermeable (SYTOX Orange, Molecular Probes) were used to assess peritoneal NET formation. Images of NETs were acquired using a FV3000 1 × 81 confocal microscope and FluoView software (Olympus) with 20x and 60x objectives. Z-stacked images were obtained over a range of 10 μm with a 1 μm step size for each randomly selected high-power field and processed using Olympus FluoView (Olympus), Adobe Photoshop (Adobe), and ImageJ (NIH) software.
10
Quantifying Neutrophil Extracellular Trap Formation
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We evaluated mouse NET formation via live cell imaging with confocal microscopy as previously described (12 (link)). Sham mice and CLP animals from all experimental groups were euthanized by CO2 inhalation and the peritoneal fluid was collected. An aliquot of 100 μL of the peritoneal fluid was placed on glass coverslips coated with poly-L-lysine for 1 hour at 37°C in 5% CO2/95% air. NET formation was assessed using cell-permeable (SYTO Green, Molecular Probes) and cell-impermeable (SYTOX Orange, Molecular Probes) DNA fluorescent stains. Using a FV3000 1X81 confocal microscope and FluoView software (Olympus) with 20X and 60X objectives, we obtained z-series images over a range of 20 μm with a 1 μm step size for each field. We processed these images using FluoView (Olympus), Photoshop (Adobe), and ImageJ (NIH) software.
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