Incubation:Article Title: Herpes Simplex Virus Type 2 Glycoprotein H Interacts with Integrin v 3 To Facilitate Viral Entry and Calcium Signaling in Human Genital Tract Epithelial Cells
Article Snippet: .. CaSki cells (5 104) or primary vaginal cells (3 104) were seeded in 96-well black plates with clear bottoms (3340, CellBIND surface; Corning Inc., NY) and incubated with 25 M fura-2 AM diluted in PBS (F1221; Invitrogen Molecular Probes) for 60 min at 37°C, rinsed thrice with PBS, placed on ice, and then exposed to cold purified HSV-2 (MOI, 5 PFU/cell) or control buffer (PBS). .. The cells were then transferred to a SpectraMaxMFe temperature-regulated chamber at 37°C (Molecular Devices) without washing; photometric data for [Ca2 ] were generated by exciting cells at 340 and 380 nm and measuring emission at 510 nm every minute for 1 h. An intracellular calibration was performed with each experiment by determining the fluorescence ratio (340:380) in the presence of Ca2 -free 10 mM K2-EGTA buffer (Rmin) and 10 mM Ca-EGTA buffer containing 10 M ionomycin (Rmax) (C-3008, calcium calibration buffer kit no. 1; Invitrogen Molecular Probes).
Article Title: Cell-impermeable staurosporine analog targets extracellular kinases to inhibit HSV and SARS-CoV-2.
Article Snippet: .. HaCat cells (5 × 104) were seeded in 96-well black plates with clear bottoms (3340, CellBIND surface, Corning Inc., NY) and incubated with 25 μM Fura-2 AM diluted in PBS (F1221, Invitrogen Molecular Probes) for 60 min at 37 °C, rinsed with PBS thrice, placed on ice and then exposed to cold dextran gradient purified HSV-2 (5 pfu/cell) or control buffer (PBS)5. .. The cells were then transferred to SpectraMaxMFe temperature-regulated chamber at 37oC (Molecular Devices Ca) without washing; photometric data for intracellular Ca2+ concentration [Ca2+] were generated by exciting cells at 340 and 380 nm and measuring emission at 510 nm every minute for one hour using SoftMaxPro.
Article Title: Herpes Simplex Virus Type 2 Glycoprotein H Interacts with Integrin αvβ3 To Facilitate Viral Entry and Calcium Signaling in Human Genital Tract Epithelial Cells
Article Snippet: .. CaSki cells (5 × 10 4 ) or primary vaginal cells (3 × 10 4 ) were seeded in 96-well black plates with clear bottoms (3340, CellBIND surface; Corning Inc., NY) and incubated with 25 μM fura-2 AM diluted in PBS (F1221; Invitrogen Molecular Probes) for 60 min at 37°C, rinsed thrice with PBS, placed on ice, and then exposed to cold purified HSV-2 (MOI, ∼5 PFU/cell) or control buffer (PBS). .. The cells were then transferred to a SpectraMaxMF e temperature-regulated chamber at 37°C (Molecular Devices) without washing; photometric data for [Ca 2+ ] were generated by exciting cells at 340 and 380 nm and measuring emission at 510 nm every minute for 1 h. An intracellular calibration was performed with each experiment by determining the fluorescence ratio (340:380) in the presence of Ca 2+ -free 10 mM K 2 -EGTA buffer ( R min ) and 10 mM Ca-EGTA buffer containing 10 μM ionomycin ( R max ) (C-3008, calcium calibration buffer kit no. 1; Invitrogen Molecular Probes).
Article Title: Cell-impermeable staurosporine analog targets extracellular kinases to inhibit HSV and SARS-CoV-2
Article Snippet: After three washes with PBS supplemented with 1% BSA, cells were harvested, solubilized in PBS containing a proteinase inhibitor cocktail, precipitated with streptavidin magnetic beads (Dynabeads M-280 Streptavidin; Life Technologies, Gaithersburg, MD, USA) and analyzed by immunoblotting. .. HaCat cells (5 × 10 4 ) were seeded in 96-well black plates with clear bottoms (3340, CellBIND surface, Corning Inc., NY) and incubated with 25 μM Fura-2 AM diluted in PBS (F1221, Invitrogen Molecular Probes) for 60 min at 37 °C, rinsed with PBS thrice, placed on ice and then exposed to cold dextran gradient purified HSV-2 (5 pfu/cell) or control buffer (PBS) . .. The cells were then transferred to SpectraMaxMF e temperature-regulated chamber at 37 o C (Molecular Devices Ca) without washing; photometric data for intracellular Ca 2+ concentration [Ca 2+ ] were generated by exciting cells at 340 and 380 nm and measuring emission at 510 nm every minute for one hour using SoftMaxPro.
Purification:Article Title: Herpes Simplex Virus Type 2 Glycoprotein H Interacts with Integrin v 3 To Facilitate Viral Entry and Calcium Signaling in Human Genital Tract Epithelial Cells
Article Snippet: .. CaSki cells (5 104) or primary vaginal cells (3 104) were seeded in 96-well black plates with clear bottoms (3340, CellBIND surface; Corning Inc., NY) and incubated with 25 M fura-2 AM diluted in PBS (F1221; Invitrogen Molecular Probes) for 60 min at 37°C, rinsed thrice with PBS, placed on ice, and then exposed to cold purified HSV-2 (MOI, 5 PFU/cell) or control buffer (PBS). .. The cells were then transferred to a SpectraMaxMFe temperature-regulated chamber at 37°C (Molecular Devices) without washing; photometric data for [Ca2 ] were generated by exciting cells at 340 and 380 nm and measuring emission at 510 nm every minute for 1 h. An intracellular calibration was performed with each experiment by determining the fluorescence ratio (340:380) in the presence of Ca2 -free 10 mM K2-EGTA buffer (Rmin) and 10 mM Ca-EGTA buffer containing 10 M ionomycin (Rmax) (C-3008, calcium calibration buffer kit no. 1; Invitrogen Molecular Probes).
Article Title: Cell-impermeable staurosporine analog targets extracellular kinases to inhibit HSV and SARS-CoV-2.
Article Snippet: .. HaCat cells (5 × 104) were seeded in 96-well black plates with clear bottoms (3340, CellBIND surface, Corning Inc., NY) and incubated with 25 μM Fura-2 AM diluted in PBS (F1221, Invitrogen Molecular Probes) for 60 min at 37 °C, rinsed with PBS thrice, placed on ice and then exposed to cold dextran gradient purified HSV-2 (5 pfu/cell) or control buffer (PBS)5. .. The cells were then transferred to SpectraMaxMFe temperature-regulated chamber at 37oC (Molecular Devices Ca) without washing; photometric data for intracellular Ca2+ concentration [Ca2+] were generated by exciting cells at 340 and 380 nm and measuring emission at 510 nm every minute for one hour using SoftMaxPro.
Article Title: Herpes Simplex Virus Type 2 Glycoprotein H Interacts with Integrin αvβ3 To Facilitate Viral Entry and Calcium Signaling in Human Genital Tract Epithelial Cells
Article Snippet: .. CaSki cells (5 × 10 4 ) or primary vaginal cells (3 × 10 4 ) were seeded in 96-well black plates with clear bottoms (3340, CellBIND surface; Corning Inc., NY) and incubated with 25 μM fura-2 AM diluted in PBS (F1221; Invitrogen Molecular Probes) for 60 min at 37°C, rinsed thrice with PBS, placed on ice, and then exposed to cold purified HSV-2 (MOI, ∼5 PFU/cell) or control buffer (PBS). .. The cells were then transferred to a SpectraMaxMF e temperature-regulated chamber at 37°C (Molecular Devices) without washing; photometric data for [Ca 2+ ] were generated by exciting cells at 340 and 380 nm and measuring emission at 510 nm every minute for 1 h. An intracellular calibration was performed with each experiment by determining the fluorescence ratio (340:380) in the presence of Ca 2+ -free 10 mM K 2 -EGTA buffer ( R min ) and 10 mM Ca-EGTA buffer containing 10 μM ionomycin ( R max ) (C-3008, calcium calibration buffer kit no. 1; Invitrogen Molecular Probes).
Article Title: Cell-impermeable staurosporine analog targets extracellular kinases to inhibit HSV and SARS-CoV-2
Article Snippet: After three washes with PBS supplemented with 1% BSA, cells were harvested, solubilized in PBS containing a proteinase inhibitor cocktail, precipitated with streptavidin magnetic beads (Dynabeads M-280 Streptavidin; Life Technologies, Gaithersburg, MD, USA) and analyzed by immunoblotting. .. HaCat cells (5 × 10 4 ) were seeded in 96-well black plates with clear bottoms (3340, CellBIND surface, Corning Inc., NY) and incubated with 25 μM Fura-2 AM diluted in PBS (F1221, Invitrogen Molecular Probes) for 60 min at 37 °C, rinsed with PBS thrice, placed on ice and then exposed to cold dextran gradient purified HSV-2 (5 pfu/cell) or control buffer (PBS) . .. The cells were then transferred to SpectraMaxMF e temperature-regulated chamber at 37 o C (Molecular Devices Ca) without washing; photometric data for intracellular Ca 2+ concentration [Ca 2+ ] were generated by exciting cells at 340 and 380 nm and measuring emission at 510 nm every minute for one hour using SoftMaxPro.
Control:Article Title: Herpes Simplex Virus Type 2 Glycoprotein H Interacts with Integrin v 3 To Facilitate Viral Entry and Calcium Signaling in Human Genital Tract Epithelial Cells
Article Snippet: .. CaSki cells (5 104) or primary vaginal cells (3 104) were seeded in 96-well black plates with clear bottoms (3340, CellBIND surface; Corning Inc., NY) and incubated with 25 M fura-2 AM diluted in PBS (F1221; Invitrogen Molecular Probes) for 60 min at 37°C, rinsed thrice with PBS, placed on ice, and then exposed to cold purified HSV-2 (MOI, 5 PFU/cell) or control buffer (PBS). .. The cells were then transferred to a SpectraMaxMFe temperature-regulated chamber at 37°C (Molecular Devices) without washing; photometric data for [Ca2 ] were generated by exciting cells at 340 and 380 nm and measuring emission at 510 nm every minute for 1 h. An intracellular calibration was performed with each experiment by determining the fluorescence ratio (340:380) in the presence of Ca2 -free 10 mM K2-EGTA buffer (Rmin) and 10 mM Ca-EGTA buffer containing 10 M ionomycin (Rmax) (C-3008, calcium calibration buffer kit no. 1; Invitrogen Molecular Probes).
Article Title: Cell-impermeable staurosporine analog targets extracellular kinases to inhibit HSV and SARS-CoV-2.
Article Snippet: .. HaCat cells (5 × 104) were seeded in 96-well black plates with clear bottoms (3340, CellBIND surface, Corning Inc., NY) and incubated with 25 μM Fura-2 AM diluted in PBS (F1221, Invitrogen Molecular Probes) for 60 min at 37 °C, rinsed with PBS thrice, placed on ice and then exposed to cold dextran gradient purified HSV-2 (5 pfu/cell) or control buffer (PBS)5. .. The cells were then transferred to SpectraMaxMFe temperature-regulated chamber at 37oC (Molecular Devices Ca) without washing; photometric data for intracellular Ca2+ concentration [Ca2+] were generated by exciting cells at 340 and 380 nm and measuring emission at 510 nm every minute for one hour using SoftMaxPro.
Article Title: Herpes Simplex Virus Type 2 Glycoprotein H Interacts with Integrin αvβ3 To Facilitate Viral Entry and Calcium Signaling in Human Genital Tract Epithelial Cells
Article Snippet: .. CaSki cells (5 × 10 4 ) or primary vaginal cells (3 × 10 4 ) were seeded in 96-well black plates with clear bottoms (3340, CellBIND surface; Corning Inc., NY) and incubated with 25 μM fura-2 AM diluted in PBS (F1221; Invitrogen Molecular Probes) for 60 min at 37°C, rinsed thrice with PBS, placed on ice, and then exposed to cold purified HSV-2 (MOI, ∼5 PFU/cell) or control buffer (PBS). .. The cells were then transferred to a SpectraMaxMF e temperature-regulated chamber at 37°C (Molecular Devices) without washing; photometric data for [Ca 2+ ] were generated by exciting cells at 340 and 380 nm and measuring emission at 510 nm every minute for 1 h. An intracellular calibration was performed with each experiment by determining the fluorescence ratio (340:380) in the presence of Ca 2+ -free 10 mM K 2 -EGTA buffer ( R min ) and 10 mM Ca-EGTA buffer containing 10 μM ionomycin ( R max ) (C-3008, calcium calibration buffer kit no. 1; Invitrogen Molecular Probes).
Article Title: Cell-impermeable staurosporine analog targets extracellular kinases to inhibit HSV and SARS-CoV-2
Article Snippet: After three washes with PBS supplemented with 1% BSA, cells were harvested, solubilized in PBS containing a proteinase inhibitor cocktail, precipitated with streptavidin magnetic beads (Dynabeads M-280 Streptavidin; Life Technologies, Gaithersburg, MD, USA) and analyzed by immunoblotting. .. HaCat cells (5 × 10 4 ) were seeded in 96-well black plates with clear bottoms (3340, CellBIND surface, Corning Inc., NY) and incubated with 25 μM Fura-2 AM diluted in PBS (F1221, Invitrogen Molecular Probes) for 60 min at 37 °C, rinsed with PBS thrice, placed on ice and then exposed to cold dextran gradient purified HSV-2 (5 pfu/cell) or control buffer (PBS) . .. The cells were then transferred to SpectraMaxMF e temperature-regulated chamber at 37 o C (Molecular Devices Ca) without washing; photometric data for intracellular Ca 2+ concentration [Ca 2+ ] were generated by exciting cells at 340 and 380 nm and measuring emission at 510 nm every minute for one hour using SoftMaxPro.
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