Diffusion-based Assay:Article Title: Computational Design and Glycoengineering of Interferon-Lambda for Nasal Prophylaxis Against Respiratory Viruses.
Article Snippet: Z-stack images were captured, and the fluorescence intensity across various focal planes was quantified using Zen Blue software (Carl Zeiss). .. HEC Gel Diffusion Assay; Trans-Well Model: For the HEC gel diffusion assay in a trans-well system, HNEpC cells (#C-12620, PromoCell) in a 24- well microplate were starved in basal medium for 12 h prior to stimulation. .. A 500 μm-thick layer of 1.2% HEC solution was added to the trans-well inserts (#36624, SPL Life Sciences) and placed in 24-well microplates. hIFNλ3 variants (50 ng/5 μL) were loaded to the top of the gel in the insert and removed after 15 min incubation.
Article Title: Computational Design and Glycoengineering of Interferon‐Lambda for Nasal Prophylaxis Against Respiratory Viruses
Article Snippet: Z‐stack images were captured, and the fluorescence intensity across various focal planes was quantified using Zen Blue software (Carl Zeiss). .. For the HEC gel diffusion assay in a trans‐well system, HNEpC cells (#C‐12620, PromoCell) in a 24‐well microplate were starved in basal medium for 12 h prior to stimulation. .. A 500 μm‐thick layer of 1.2% HEC solution was added to the trans‐well inserts (#36624, SPL Life Sciences) and placed in 24‐well microplates. hIFN‐λ3 variants (50 ng/5 μL) were loaded to the top of the gel in the insert and removed after 15 min incubation.
Cell Culture:Article Title: Enhancing the protein fitness of interferon-lambda through computational design and glyco-engineering for prophylactic nasal drugs against respiratory viruses
Article Snippet: .. HNEpC cells (C-12620, PromoCell) were cultured in Airway Epithelial Cell Growth Medium (C-21060, PromoCell) at 37°C in a humidified 5% CO2 incubator. .. Expi 293-F cells (A14527, Thermo Fisher Scientific) were maintained in Expi293 Expression Medium (A1435102, Thermo Fisher Scientific) under shaking conditions at 37°C in a humidified 8% CO 2 incubator.
Article Title: Computational Design and Glycoengineering of Interferon-Lambda for Nasal Prophylaxis Against Respiratory Viruses.
Article Snippet: The peak fractions were pooled and concentrated to ≈1mgmL−1 using an Amicon Ultra centrifugal filter (#UFC8010, Millipore). .. For deglycosylation analysis, hIFN-λs were incubated with GSTPNGaseF (10 μg mL−1) at 37 °C for 3 h. Immunoblotting of Intracellular Signal Activation in HNEpCs: HNEpC cells (#C-12620, PromoCell) were cultured in basal Airway Epithelial cell Growth Medium (#C-21060, PromoCell) without supplement mix for starvation at 37 °C in a humidified 5% CO2 incubator for 12 h. After starvation, the cells were incubated with 100 ngmL−1 of recombinant hIFN-λs for 1 h. Cells were then washed with cold PBS and lysed with lysis buffer (10 mm Tris-Cl pH 7.4, 150 mm NaCl, 5 mm EDTA, 10% glycerol, 1% Triton X-100, protease inhibitor, phosphatase inhibitor). ..
Article Title: Enhancing the protein fitness of interferon-lambda through computational design and glyco-engineering for prophylactic nasal drugs against respiratory viruses
Article Snippet: .. HNEpC cells (#C-12620, PromoCell) were cultured in basal Airway Epithelial cell Growth Medium (#C-21060, PromoCell) without supplement mix for starvation at 37°C in a humidified 5% CO 2 incubator for 12 h. After starvation, the cells were incubated with 100 ng/mL of recombinant hIFN-λs for 1 h. Cells were then washed with cold PBS and lysed with lysis buffer (10 mM Tris-Cl pH 7.4, 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Triton X-100, protease inhibitor, phosphatase inhibitor). ..
Article Title: Computational Design and Glycoengineering of Interferon‐Lambda for Nasal Prophylaxis Against Respiratory Viruses
Article Snippet: .. HNEpC cells (#C‐12620, PromoCell) were cultured in basal Airway Epithelial cell Growth Medium (#C‐21060, PromoCell) without supplement mix for starvation at 37 °C in a humidified 5% CO 2 incubator for 12 h. After starvation, the cells were incubated with 100 ng mL −1 of recombinant hIFN‐λs for 1 h. Cells were then washed with cold PBS and lysed with lysis buffer (10 m m Tris‐Cl pH 7.4, 150 m m NaCl, 5 m m EDTA, 10% glycerol, 1% Triton X‐100, protease inhibitor, phosphatase inhibitor). ..
Incubation:Article Title: Computational Design and Glycoengineering of Interferon-Lambda for Nasal Prophylaxis Against Respiratory Viruses.
Article Snippet: The peak fractions were pooled and concentrated to ≈1mgmL−1 using an Amicon Ultra centrifugal filter (#UFC8010, Millipore). .. For deglycosylation analysis, hIFN-λs were incubated with GSTPNGaseF (10 μg mL−1) at 37 °C for 3 h. Immunoblotting of Intracellular Signal Activation in HNEpCs: HNEpC cells (#C-12620, PromoCell) were cultured in basal Airway Epithelial cell Growth Medium (#C-21060, PromoCell) without supplement mix for starvation at 37 °C in a humidified 5% CO2 incubator for 12 h. After starvation, the cells were incubated with 100 ngmL−1 of recombinant hIFN-λs for 1 h. Cells were then washed with cold PBS and lysed with lysis buffer (10 mm Tris-Cl pH 7.4, 150 mm NaCl, 5 mm EDTA, 10% glycerol, 1% Triton X-100, protease inhibitor, phosphatase inhibitor). ..
Article Title: Enhancing the protein fitness of interferon-lambda through computational design and glyco-engineering for prophylactic nasal drugs against respiratory viruses
Article Snippet: .. HNEpC cells (#C-12620, PromoCell) were cultured in basal Airway Epithelial cell Growth Medium (#C-21060, PromoCell) without supplement mix for starvation at 37°C in a humidified 5% CO 2 incubator for 12 h. After starvation, the cells were incubated with 100 ng/mL of recombinant hIFN-λs for 1 h. Cells were then washed with cold PBS and lysed with lysis buffer (10 mM Tris-Cl pH 7.4, 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Triton X-100, protease inhibitor, phosphatase inhibitor). ..
Article Title: Computational Design and Glycoengineering of Interferon‐Lambda for Nasal Prophylaxis Against Respiratory Viruses
Article Snippet: .. HNEpC cells (#C‐12620, PromoCell) were cultured in basal Airway Epithelial cell Growth Medium (#C‐21060, PromoCell) without supplement mix for starvation at 37 °C in a humidified 5% CO 2 incubator for 12 h. After starvation, the cells were incubated with 100 ng mL −1 of recombinant hIFN‐λs for 1 h. Cells were then washed with cold PBS and lysed with lysis buffer (10 m m Tris‐Cl pH 7.4, 150 m m NaCl, 5 m m EDTA, 10% glycerol, 1% Triton X‐100, protease inhibitor, phosphatase inhibitor). ..
Western Blot:Article Title: Computational Design and Glycoengineering of Interferon-Lambda for Nasal Prophylaxis Against Respiratory Viruses.
Article Snippet: The peak fractions were pooled and concentrated to ≈1mgmL−1 using an Amicon Ultra centrifugal filter (#UFC8010, Millipore). .. For deglycosylation analysis, hIFN-λs were incubated with GSTPNGaseF (10 μg mL−1) at 37 °C for 3 h. Immunoblotting of Intracellular Signal Activation in HNEpCs: HNEpC cells (#C-12620, PromoCell) were cultured in basal Airway Epithelial cell Growth Medium (#C-21060, PromoCell) without supplement mix for starvation at 37 °C in a humidified 5% CO2 incubator for 12 h. After starvation, the cells were incubated with 100 ngmL−1 of recombinant hIFN-λs for 1 h. Cells were then washed with cold PBS and lysed with lysis buffer (10 mm Tris-Cl pH 7.4, 150 mm NaCl, 5 mm EDTA, 10% glycerol, 1% Triton X-100, protease inhibitor, phosphatase inhibitor). ..
Activation Assay:Article Title: Computational Design and Glycoengineering of Interferon-Lambda for Nasal Prophylaxis Against Respiratory Viruses.
Article Snippet: The peak fractions were pooled and concentrated to ≈1mgmL−1 using an Amicon Ultra centrifugal filter (#UFC8010, Millipore). .. For deglycosylation analysis, hIFN-λs were incubated with GSTPNGaseF (10 μg mL−1) at 37 °C for 3 h. Immunoblotting of Intracellular Signal Activation in HNEpCs: HNEpC cells (#C-12620, PromoCell) were cultured in basal Airway Epithelial cell Growth Medium (#C-21060, PromoCell) without supplement mix for starvation at 37 °C in a humidified 5% CO2 incubator for 12 h. After starvation, the cells were incubated with 100 ngmL−1 of recombinant hIFN-λs for 1 h. Cells were then washed with cold PBS and lysed with lysis buffer (10 mm Tris-Cl pH 7.4, 150 mm NaCl, 5 mm EDTA, 10% glycerol, 1% Triton X-100, protease inhibitor, phosphatase inhibitor). ..
Recombinant:Article Title: Computational Design and Glycoengineering of Interferon-Lambda for Nasal Prophylaxis Against Respiratory Viruses.
Article Snippet: The peak fractions were pooled and concentrated to ≈1mgmL−1 using an Amicon Ultra centrifugal filter (#UFC8010, Millipore). .. For deglycosylation analysis, hIFN-λs were incubated with GSTPNGaseF (10 μg mL−1) at 37 °C for 3 h. Immunoblotting of Intracellular Signal Activation in HNEpCs: HNEpC cells (#C-12620, PromoCell) were cultured in basal Airway Epithelial cell Growth Medium (#C-21060, PromoCell) without supplement mix for starvation at 37 °C in a humidified 5% CO2 incubator for 12 h. After starvation, the cells were incubated with 100 ngmL−1 of recombinant hIFN-λs for 1 h. Cells were then washed with cold PBS and lysed with lysis buffer (10 mm Tris-Cl pH 7.4, 150 mm NaCl, 5 mm EDTA, 10% glycerol, 1% Triton X-100, protease inhibitor, phosphatase inhibitor). ..
Article Title: Enhancing the protein fitness of interferon-lambda through computational design and glyco-engineering for prophylactic nasal drugs against respiratory viruses
Article Snippet: .. HNEpC cells (#C-12620, PromoCell) were cultured in basal Airway Epithelial cell Growth Medium (#C-21060, PromoCell) without supplement mix for starvation at 37°C in a humidified 5% CO 2 incubator for 12 h. After starvation, the cells were incubated with 100 ng/mL of recombinant hIFN-λs for 1 h. Cells were then washed with cold PBS and lysed with lysis buffer (10 mM Tris-Cl pH 7.4, 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Triton X-100, protease inhibitor, phosphatase inhibitor). ..
Article Title: Computational Design and Glycoengineering of Interferon‐Lambda for Nasal Prophylaxis Against Respiratory Viruses
Article Snippet: .. HNEpC cells (#C‐12620, PromoCell) were cultured in basal Airway Epithelial cell Growth Medium (#C‐21060, PromoCell) without supplement mix for starvation at 37 °C in a humidified 5% CO 2 incubator for 12 h. After starvation, the cells were incubated with 100 ng mL −1 of recombinant hIFN‐λs for 1 h. Cells were then washed with cold PBS and lysed with lysis buffer (10 m m Tris‐Cl pH 7.4, 150 m m NaCl, 5 m m EDTA, 10% glycerol, 1% Triton X‐100, protease inhibitor, phosphatase inhibitor). ..
Lysis:Article Title: Computational Design and Glycoengineering of Interferon-Lambda for Nasal Prophylaxis Against Respiratory Viruses.
Article Snippet: The peak fractions were pooled and concentrated to ≈1mgmL−1 using an Amicon Ultra centrifugal filter (#UFC8010, Millipore). .. For deglycosylation analysis, hIFN-λs were incubated with GSTPNGaseF (10 μg mL−1) at 37 °C for 3 h. Immunoblotting of Intracellular Signal Activation in HNEpCs: HNEpC cells (#C-12620, PromoCell) were cultured in basal Airway Epithelial cell Growth Medium (#C-21060, PromoCell) without supplement mix for starvation at 37 °C in a humidified 5% CO2 incubator for 12 h. After starvation, the cells were incubated with 100 ngmL−1 of recombinant hIFN-λs for 1 h. Cells were then washed with cold PBS and lysed with lysis buffer (10 mm Tris-Cl pH 7.4, 150 mm NaCl, 5 mm EDTA, 10% glycerol, 1% Triton X-100, protease inhibitor, phosphatase inhibitor). ..
Article Title: Enhancing the protein fitness of interferon-lambda through computational design and glyco-engineering for prophylactic nasal drugs against respiratory viruses
Article Snippet: .. HNEpC cells (#C-12620, PromoCell) were cultured in basal Airway Epithelial cell Growth Medium (#C-21060, PromoCell) without supplement mix for starvation at 37°C in a humidified 5% CO 2 incubator for 12 h. After starvation, the cells were incubated with 100 ng/mL of recombinant hIFN-λs for 1 h. Cells were then washed with cold PBS and lysed with lysis buffer (10 mM Tris-Cl pH 7.4, 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Triton X-100, protease inhibitor, phosphatase inhibitor). ..
Article Title: Computational Design and Glycoengineering of Interferon‐Lambda for Nasal Prophylaxis Against Respiratory Viruses
Article Snippet: .. HNEpC cells (#C‐12620, PromoCell) were cultured in basal Airway Epithelial cell Growth Medium (#C‐21060, PromoCell) without supplement mix for starvation at 37 °C in a humidified 5% CO 2 incubator for 12 h. After starvation, the cells were incubated with 100 ng mL −1 of recombinant hIFN‐λs for 1 h. Cells were then washed with cold PBS and lysed with lysis buffer (10 m m Tris‐Cl pH 7.4, 150 m m NaCl, 5 m m EDTA, 10% glycerol, 1% Triton X‐100, protease inhibitor, phosphatase inhibitor). ..
Protease Inhibitor:Article Title: Computational Design and Glycoengineering of Interferon-Lambda for Nasal Prophylaxis Against Respiratory Viruses.
Article Snippet: The peak fractions were pooled and concentrated to ≈1mgmL−1 using an Amicon Ultra centrifugal filter (#UFC8010, Millipore). .. For deglycosylation analysis, hIFN-λs were incubated with GSTPNGaseF (10 μg mL−1) at 37 °C for 3 h. Immunoblotting of Intracellular Signal Activation in HNEpCs: HNEpC cells (#C-12620, PromoCell) were cultured in basal Airway Epithelial cell Growth Medium (#C-21060, PromoCell) without supplement mix for starvation at 37 °C in a humidified 5% CO2 incubator for 12 h. After starvation, the cells were incubated with 100 ngmL−1 of recombinant hIFN-λs for 1 h. Cells were then washed with cold PBS and lysed with lysis buffer (10 mm Tris-Cl pH 7.4, 150 mm NaCl, 5 mm EDTA, 10% glycerol, 1% Triton X-100, protease inhibitor, phosphatase inhibitor). ..
Article Title: Enhancing the protein fitness of interferon-lambda through computational design and glyco-engineering for prophylactic nasal drugs against respiratory viruses
Article Snippet: .. HNEpC cells (#C-12620, PromoCell) were cultured in basal Airway Epithelial cell Growth Medium (#C-21060, PromoCell) without supplement mix for starvation at 37°C in a humidified 5% CO 2 incubator for 12 h. After starvation, the cells were incubated with 100 ng/mL of recombinant hIFN-λs for 1 h. Cells were then washed with cold PBS and lysed with lysis buffer (10 mM Tris-Cl pH 7.4, 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Triton X-100, protease inhibitor, phosphatase inhibitor). ..
Article Title: Computational Design and Glycoengineering of Interferon‐Lambda for Nasal Prophylaxis Against Respiratory Viruses
Article Snippet: .. HNEpC cells (#C‐12620, PromoCell) were cultured in basal Airway Epithelial cell Growth Medium (#C‐21060, PromoCell) without supplement mix for starvation at 37 °C in a humidified 5% CO 2 incubator for 12 h. After starvation, the cells were incubated with 100 ng mL −1 of recombinant hIFN‐λs for 1 h. Cells were then washed with cold PBS and lysed with lysis buffer (10 m m Tris‐Cl pH 7.4, 150 m m NaCl, 5 m m EDTA, 10% glycerol, 1% Triton X‐100, protease inhibitor, phosphatase inhibitor). ..
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