plasmid encoding the envelope of vesicular stomatitis virus (vsvg) (pmd.2g)  (Addgene inc)

Journal: STAR Protocols

Article Title: Protocol for intracellular immunofluorescence measurements of latent HIV reactivation in a primary CD4 + T cell model

doi: 10.1016/j.xpro.2024.103398

Figure Lengend Snippet: Monitoring the generation of latently infected Th17 cells upon infection with VSVG-pseudotyped packaged virus of the d2EGFP single-round viral construct Top panel , Dual assessment of the percentage of EGFP-positive cells, which indicates the extent of proviral gene expression, and their cell viability based on propidium iodide (PI) staining. Bottom panel, Corresponding forward/side scatter plots. pi : post-infection; ps : post-shutdown.

Article Snippet: pMD.G VSVG , Addgene , #8454.

Techniques: Infection, Virus, Construct, Gene Expression, Staining

Journal: STAR Protocols

Article Title: Protocol for intracellular immunofluorescence measurements of latent HIV reactivation in a primary CD4 + T cell model

doi: 10.1016/j.xpro.2024.103398

Figure Lengend Snippet: Tat/Nef dual immunofluorescence flow cytometry of cells infected with VSVG-pseudotyped virus of the d2EGFP single-round viral construct Uninfected, acutely infected, latently infected, and reactivated Th17 cells were immunostained for Tat and Nef as described in the text. The 2D plots show proviral gene expression as monitored by d2EGFP versus Tat (Top) , d2EGFP versus Nef (Middle), and Tat versus Nef (Bottom) .

Article Snippet: pMD.G VSVG , Addgene , #8454.

Techniques: Immunofluorescence, Flow Cytometry, Infection, Virus, Construct, Gene Expression

Journal: STAR Protocols

Article Title: Protocol for intracellular immunofluorescence measurements of latent HIV reactivation in a primary CD4 + T cell model

doi: 10.1016/j.xpro.2024.103398

Figure Lengend Snippet: Infection of polarized Th17 cells with VSVG-pseudotyped virus of the CD8a-d2EGFP single-round viral construct and their subsequent positive selection by anti-CD8a sorting In this experiment, cells were CD8a-selected at Day 5 post-infection (pi). CD8a-d2EGFP is relatively stable and remains expressed even in the quiescent, latently infected cells.

Article Snippet: pMD.G VSVG , Addgene , #8454.

Techniques: Infection, Virus, Construct, Selection

Journal: STAR Protocols

Article Title: Protocol for intracellular immunofluorescence measurements of latent HIV reactivation in a primary CD4 + T cell model

doi: 10.1016/j.xpro.2024.103398

Figure Lengend Snippet:

Article Snippet: pMD.G VSVG , Addgene , #8454.

Techniques: Blocking Assay, Recombinant, Cell Isolation, Selection, Antibody Labeling

Journal: STAR protocols

Article Title: Protocol for intracellular immunofluorescence measurements of latent HIV reactivation in a primary CD4 + T cell model.

doi: 10.1016/j.xpro.2024.103398

Figure Lengend Snippet: Figure 2. Monitoring the generation of latently infected Th17 cells upon infection with VSVG-pseudotyped packaged virus of the d2EGFP single- round viral construct Top panel, Dual assessment of the percentage of EGFP-positive cells, which indicates the extent of proviral gene expression, and their cell viability based on propidium iodide (PI) staining. Bottom panel, Corresponding forward/side scatter plots. pi: post-infection; ps: post-shutdown.

Article Snippet: Continued REAGENT or RESOURCE SOURCE IDENTIFIER IL-7 PeproTech 200-07 IL-8 PeproTech 200-08M 1L-10 PeproTech 200-10 IL-23 PeproTech 200-23 TGF-b PeproTech 100-21C Anti-human IL-4 PeproTech 500-M04 Anti-human IFN-g PeproTech 500-M90 Concanavalin A MilliporeSigma C0412 Critical commercial assays Human CD4 naive T cell isolation kit BioLegend 480042 Mouse CD8a positive selection kit STEMCELL 18953 Pacific Blue antibody labeling kit Thermo Fisher Scientific P30013 Alexa Fluor 488 antibody labeling kit Thermo Fisher Scientific A20181 Alexa Fluor 555 antibody labeling kit Thermo Fisher Scientific A20187 Alexa Fluor 647 antibody labeling kit Thermo Fisher Scientific A20186 Experimental models: cell lines HEK293T ATCC CRL-3216 Experimental models: Organisms/strains Polarized and expanded primary Th17 cells This paper N/A Recombinant DNA pHR’-d2EGFP-Nef+ Dobrowolski et al.2 Dobrowolski et al.2 pHR’-CD8a-d2EGFP-Nef+ Dobrowolski et al.2 Dobrowolski et al.2 Lentiviral gag/pol pCMV-Delta 8.91r Dobrowolski et al.2 Dobrowolski et al.2 pMD.G VSVG Addgene #8454 Other Optima L-100K ultracentrifuge Beckman Coulter N/A SW32 Ti rotor Beckman Coulter N/A BD LSR Fortessa BD Biosciences N/A EasySep magnetic tube holder STEMCELL 18000 Eppendorf 5810R benchtop centrifuge Eppendorf 022625101 STAR Protocols 5, 103398, December 20, 2024 3 Day 2: Generation of single-round VSVG-pseudotyped HIV 2.

Techniques: Infection, Virus, Construct, Gene Expression, Staining

Journal: STAR protocols

Article Title: Protocol for intracellular immunofluorescence measurements of latent HIV reactivation in a primary CD4 + T cell model.

doi: 10.1016/j.xpro.2024.103398

Figure Lengend Snippet: Figure 3. Tat/Nef dual immunofluorescence flow cytometry of cells infected with VSVG-pseudotyped virus of the d2EGFP single-round viral construct Uninfected, acutely infected, latently infected, and reactivated Th17 cells were immunostained for Tat and Nef as described in the text. The 2D plots show proviral gene expression as monitored by d2EGFP versus Tat (Top), d2EGFP versus Nef (Middle), and Tat versus Nef (Bottom).

Article Snippet: Continued REAGENT or RESOURCE SOURCE IDENTIFIER IL-7 PeproTech 200-07 IL-8 PeproTech 200-08M 1L-10 PeproTech 200-10 IL-23 PeproTech 200-23 TGF-b PeproTech 100-21C Anti-human IL-4 PeproTech 500-M04 Anti-human IFN-g PeproTech 500-M90 Concanavalin A MilliporeSigma C0412 Critical commercial assays Human CD4 naive T cell isolation kit BioLegend 480042 Mouse CD8a positive selection kit STEMCELL 18953 Pacific Blue antibody labeling kit Thermo Fisher Scientific P30013 Alexa Fluor 488 antibody labeling kit Thermo Fisher Scientific A20181 Alexa Fluor 555 antibody labeling kit Thermo Fisher Scientific A20187 Alexa Fluor 647 antibody labeling kit Thermo Fisher Scientific A20186 Experimental models: cell lines HEK293T ATCC CRL-3216 Experimental models: Organisms/strains Polarized and expanded primary Th17 cells This paper N/A Recombinant DNA pHR’-d2EGFP-Nef+ Dobrowolski et al.2 Dobrowolski et al.2 pHR’-CD8a-d2EGFP-Nef+ Dobrowolski et al.2 Dobrowolski et al.2 Lentiviral gag/pol pCMV-Delta 8.91r Dobrowolski et al.2 Dobrowolski et al.2 pMD.G VSVG Addgene #8454 Other Optima L-100K ultracentrifuge Beckman Coulter N/A SW32 Ti rotor Beckman Coulter N/A BD LSR Fortessa BD Biosciences N/A EasySep magnetic tube holder STEMCELL 18000 Eppendorf 5810R benchtop centrifuge Eppendorf 022625101 STAR Protocols 5, 103398, December 20, 2024 3 Day 2: Generation of single-round VSVG-pseudotyped HIV 2.

Techniques: Cytometry, Infection, Virus, Construct, Gene Expression

Journal: STAR protocols

Article Title: Protocol for intracellular immunofluorescence measurements of latent HIV reactivation in a primary CD4 + T cell model.

doi: 10.1016/j.xpro.2024.103398

Figure Lengend Snippet: Figure 4. Infection of polarized Th17 cells with VSVG-pseudotyped virus of the CD8a-d2EGFP single-round viral construct and their subsequent positive selection by anti-CD8a sorting In this experiment, cells were CD8a-selected at Day 5 post-infection (pi). CD8a-d2EGFP is relatively stable and remains expressed even in the quiescent, latently infected cells.

Article Snippet: Continued REAGENT or RESOURCE SOURCE IDENTIFIER IL-7 PeproTech 200-07 IL-8 PeproTech 200-08M 1L-10 PeproTech 200-10 IL-23 PeproTech 200-23 TGF-b PeproTech 100-21C Anti-human IL-4 PeproTech 500-M04 Anti-human IFN-g PeproTech 500-M90 Concanavalin A MilliporeSigma C0412 Critical commercial assays Human CD4 naive T cell isolation kit BioLegend 480042 Mouse CD8a positive selection kit STEMCELL 18953 Pacific Blue antibody labeling kit Thermo Fisher Scientific P30013 Alexa Fluor 488 antibody labeling kit Thermo Fisher Scientific A20181 Alexa Fluor 555 antibody labeling kit Thermo Fisher Scientific A20187 Alexa Fluor 647 antibody labeling kit Thermo Fisher Scientific A20186 Experimental models: cell lines HEK293T ATCC CRL-3216 Experimental models: Organisms/strains Polarized and expanded primary Th17 cells This paper N/A Recombinant DNA pHR’-d2EGFP-Nef+ Dobrowolski et al.2 Dobrowolski et al.2 pHR’-CD8a-d2EGFP-Nef+ Dobrowolski et al.2 Dobrowolski et al.2 Lentiviral gag/pol pCMV-Delta 8.91r Dobrowolski et al.2 Dobrowolski et al.2 pMD.G VSVG Addgene #8454 Other Optima L-100K ultracentrifuge Beckman Coulter N/A SW32 Ti rotor Beckman Coulter N/A BD LSR Fortessa BD Biosciences N/A EasySep magnetic tube holder STEMCELL 18000 Eppendorf 5810R benchtop centrifuge Eppendorf 022625101 STAR Protocols 5, 103398, December 20, 2024 3 Day 2: Generation of single-round VSVG-pseudotyped HIV 2.

Techniques: Infection, Virus, Construct, Selection