Imaging:Article Title: Human transbodies that interfere with the functions of Ebola virus VP35 protein in genome replication and transcription and innate immune antagonism
Article Snippet: .. HepG2 cells (6 × 10 4 cells) were placed in an 8-chamber cell imaging cover-glass (Eppendorf, Hamburg, Germany) and kept at 37 °C in a 5% CO 2 incubator overnight. .. The culture medium was removed, and the cells were replenished with 0.5 mL fresh complete medium containing 25 μg/mL of individual R9-HuscFvs and then further incubated for 3 h. The cells were gently rinsed with Dulbecco’s PBS (DPBS) (Gibco), fixed with a 1:2 dilution of IC fixation buffer (eBioscience, Thermo Fisher Scientific) in DPBS at 25 °C for 1 h, washed with PBS, permeabilized using 0.1% v/v Triton X-100 in PBS at 37 °C for 30 min, and blocked with blocking solution (1% w/v BSA, 22.52 mg/mL glycine, and 0.1% v/v Tween-20 in PBS) at 25 °C for 1 h. After blocking, the cells were washed with PBS and stained with StrepMAB ImmoChromeo 488-conjugated antibody (IBA Life Sciences GmbH) and 1 μg/mL Hoechst 33342 (Biotium, Fremont, CA, USA) in blocking solution at 4 °C overnight.
Article Title: Activating PKC-ε induces HIV expression with improved tolerability.
Article Snippet: .. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1012874 February 6, 2025 23 / 31 Confocal microscopy to study translocation of PKC isoforms A549 cells expressing tGFP-labeled PKC isoforms were plated in an 8-chamber cell imaging cover glass (Eppendorf, catalog 0030742036) and cultured in RPMI medium supplemented with 10% heat-inactivated FBS, antibiotic-antimycotic agent (Gibco-BRL, catalog 15240-062) antimycoplasma agent, Plasmocin (InvivoGen, catalog ant-mpt-1). .. Cells were pretreated with Hoechst 3342 for nuclear staining and then imaged upon addition of the compounds at 37oC to assess localization of tGFP-labeled proteins using confocal laser scanning microscopy with a Leica SP8 (Leica Microsystems Inc., Wetzlar, Germany).
Article Title: Activating PKC-ε induces HIV expression with improved tolerability
Article Snippet: .. A549 cells expressing tGFP-labeled PKC isoforms were plated in an 8-chamber cell imaging cover glass (Eppendorf, catalog 0030742036) and cultured in RPMI medium supplemented with 10% heat-inactivated FBS, antibiotic-antimycotic agent (Gibco-BRL, catalog 15240-062) antimycoplasma agent, Plasmocin (InvivoGen, catalog ant-mpt-1). .. Cells were pretreated with Hoechst 3342 for nuclear staining and then imaged upon addition of the compounds at 37oC to assess localization of tGFP-labeled proteins using confocal laser scanning microscopy with a Leica SP8 (Leica Microsystems Inc., Wetzlar, Germany).
Article Title: Human transbodies that interfere with the functions of Ebola virus VP35 protein in genome replication and transcription and innate immune antagonism.
Article Snippet: .. HepG2 cells (6× 104 cells) were placed in an 8-chamber cell imaging cover-glass (Eppendorf, Hamburg, Germany) and kept at 37 °C in a 5% CO2 incubator overnight. .. The culture medium was removed, and the cells were replenished with 0.5 mL fresh complete medium containing 25 μg/mL of individual R9-HuscFvs and then further incubated for 3 h. The cells were gently rinsed with Dulbecco’s PBS (DPBS) (Gibco), fixed with a 1:2 dilution of IC fixation buffer (eBioscience, Thermo Fisher Scientific) in DPBS at 25 °C for 1 h, washed with PBS, permeabilized using 0.1% v/v Triton X-100 in PBS at 37 ° C for 30min, and blocked with blocking solution (1% w/v BSA, 22.52 mg/mL glycine, and 0.1% v/v Tween-20 in PBS) at 25 °C for 1 h. After blocking, the cells were washed with PBS and stained with StrepMAB ImmoChromeo 488-conjugated antibody (IBA Life Sciences GmbH) and 1 μg/mL Hoechst 33342 (Biotium, Fremont, CA, USA) in blocking solution at 4 °C overnight.
Confocal Microscopy:Article Title: Activating PKC-ε induces HIV expression with improved tolerability.
Article Snippet: .. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1012874 February 6, 2025 23 / 31 Confocal microscopy to study translocation of PKC isoforms A549 cells expressing tGFP-labeled PKC isoforms were plated in an 8-chamber cell imaging cover glass (Eppendorf, catalog 0030742036) and cultured in RPMI medium supplemented with 10% heat-inactivated FBS, antibiotic-antimycotic agent (Gibco-BRL, catalog 15240-062) antimycoplasma agent, Plasmocin (InvivoGen, catalog ant-mpt-1). .. Cells were pretreated with Hoechst 3342 for nuclear staining and then imaged upon addition of the compounds at 37oC to assess localization of tGFP-labeled proteins using confocal laser scanning microscopy with a Leica SP8 (Leica Microsystems Inc., Wetzlar, Germany).
Translocation Assay:Article Title: Activating PKC-ε induces HIV expression with improved tolerability.
Article Snippet: .. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1012874 February 6, 2025 23 / 31 Confocal microscopy to study translocation of PKC isoforms A549 cells expressing tGFP-labeled PKC isoforms were plated in an 8-chamber cell imaging cover glass (Eppendorf, catalog 0030742036) and cultured in RPMI medium supplemented with 10% heat-inactivated FBS, antibiotic-antimycotic agent (Gibco-BRL, catalog 15240-062) antimycoplasma agent, Plasmocin (InvivoGen, catalog ant-mpt-1). .. Cells were pretreated with Hoechst 3342 for nuclear staining and then imaged upon addition of the compounds at 37oC to assess localization of tGFP-labeled proteins using confocal laser scanning microscopy with a Leica SP8 (Leica Microsystems Inc., Wetzlar, Germany).
Expressing:Article Title: Activating PKC-ε induces HIV expression with improved tolerability.
Article Snippet: .. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1012874 February 6, 2025 23 / 31 Confocal microscopy to study translocation of PKC isoforms A549 cells expressing tGFP-labeled PKC isoforms were plated in an 8-chamber cell imaging cover glass (Eppendorf, catalog 0030742036) and cultured in RPMI medium supplemented with 10% heat-inactivated FBS, antibiotic-antimycotic agent (Gibco-BRL, catalog 15240-062) antimycoplasma agent, Plasmocin (InvivoGen, catalog ant-mpt-1). .. Cells were pretreated with Hoechst 3342 for nuclear staining and then imaged upon addition of the compounds at 37oC to assess localization of tGFP-labeled proteins using confocal laser scanning microscopy with a Leica SP8 (Leica Microsystems Inc., Wetzlar, Germany).
Article Title: Activating PKC-ε induces HIV expression with improved tolerability
Article Snippet: .. A549 cells expressing tGFP-labeled PKC isoforms were plated in an 8-chamber cell imaging cover glass (Eppendorf, catalog 0030742036) and cultured in RPMI medium supplemented with 10% heat-inactivated FBS, antibiotic-antimycotic agent (Gibco-BRL, catalog 15240-062) antimycoplasma agent, Plasmocin (InvivoGen, catalog ant-mpt-1). .. Cells were pretreated with Hoechst 3342 for nuclear staining and then imaged upon addition of the compounds at 37oC to assess localization of tGFP-labeled proteins using confocal laser scanning microscopy with a Leica SP8 (Leica Microsystems Inc., Wetzlar, Germany).
Cell Culture:Article Title: Activating PKC-ε induces HIV expression with improved tolerability.
Article Snippet: .. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1012874 February 6, 2025 23 / 31 Confocal microscopy to study translocation of PKC isoforms A549 cells expressing tGFP-labeled PKC isoforms were plated in an 8-chamber cell imaging cover glass (Eppendorf, catalog 0030742036) and cultured in RPMI medium supplemented with 10% heat-inactivated FBS, antibiotic-antimycotic agent (Gibco-BRL, catalog 15240-062) antimycoplasma agent, Plasmocin (InvivoGen, catalog ant-mpt-1). .. Cells were pretreated with Hoechst 3342 for nuclear staining and then imaged upon addition of the compounds at 37oC to assess localization of tGFP-labeled proteins using confocal laser scanning microscopy with a Leica SP8 (Leica Microsystems Inc., Wetzlar, Germany).
Article Title: Activating PKC-ε induces HIV expression with improved tolerability
Article Snippet: .. A549 cells expressing tGFP-labeled PKC isoforms were plated in an 8-chamber cell imaging cover glass (Eppendorf, catalog 0030742036) and cultured in RPMI medium supplemented with 10% heat-inactivated FBS, antibiotic-antimycotic agent (Gibco-BRL, catalog 15240-062) antimycoplasma agent, Plasmocin (InvivoGen, catalog ant-mpt-1). .. Cells were pretreated with Hoechst 3342 for nuclear staining and then imaged upon addition of the compounds at 37oC to assess localization of tGFP-labeled proteins using confocal laser scanning microscopy with a Leica SP8 (Leica Microsystems Inc., Wetzlar, Germany).
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