human anti-ccr5 primary antibody  (Biogenex)

Journal: Journal of Translational Medicine

Article Title: Identifying CCR5 coreceptor populations permissive for HIV-1 entry and productive infection: implications for in vivo studies

doi: 10.1186/s12967-022-03243-8

Figure Lengend Snippet: Comparison of dual staining CCR5 imaging methods. The C-terminus is GFP tagged (green); the extracellular epitopes are stained with domain specific mAbs (red) ( A ). A U87.CD4.CCR5-GFP cell expressing CCR5 C-terminus-fused GFP, stained with Alexa 647-conjugated anti-ECL1/ECL2 45523 mAb ( B ) and a confocal image under the same conditions ( C ). Bar size—10 µm

Article Snippet: Each group was stained with one of 1:10 T 21/8 (Invitrogen, Cat. 14-1957-82), CTC5 (R&D Systems, Cat. MAB1802), CTC8 (R&D Systems, Cat. MAB1801), 2D7 (BD Biosciences, Cat. 555991), 45523 (R&D Systems, Cat. MAB181), or 45531 (R&D Systems, Cat. MAB182) CCR5 primary antibodies in PBS with 1% FBSΔ for 20 min at RT, followed by 1:500 anti-mouse AlexaFluor 647 (abcam, Cat. ab150107) under the same conditions.

Techniques: Comparison, Staining, Imaging, Expressing

Journal: Journal of Translational Medicine

Article Title: Identifying CCR5 coreceptor populations permissive for HIV-1 entry and productive infection: implications for in vivo studies

doi: 10.1186/s12967-022-03243-8

Figure Lengend Snippet: Colocalization analysis of CCR5 epitopes. A simplified example of how colocalization is quantified. When the NT or ECL2 CCR5 epitope antibody (red) labels a CCR5 C-terminus-GFP fusion protein (green), the overlapping fluorophores create a yellow signal. Unlabeled ectopically expressed CCR5-GFP fusion protein will show as green only and labeled endogenously expressed CCR5 will show as red only. The color identity and density spread are displayed in the graph along with the intensity thresholds set (white lines) using single color controls. The inset is a cartoon of which category a pixel within the image is assigned based on the thresholds, with 1 being green only, 2 being red only, 3 being yellow (overlap), and 4 being background ( A ). A cell ( B ) is analyzed using the ZEN Blue 2.3 software, and each of the colors are identified by the computer based on the parameters set. The extracellular CCR5 epitope in this example (NT; CTC8) is highlighted in orange, CCR5-GFP in cyan, and NT + C-terminus-GFP overlap in dark blue by the analysis software ( C ). The pixels in these identified regions are counted by the program, and the colocalization coefficient is calculated (dark blue/dark blue + cyan) to determine what percentage of CCR5-GFP was bound by the antibody. Bar size—5 µm

Article Snippet: Each group was stained with one of 1:10 T 21/8 (Invitrogen, Cat. 14-1957-82), CTC5 (R&D Systems, Cat. MAB1802), CTC8 (R&D Systems, Cat. MAB1801), 2D7 (BD Biosciences, Cat. 555991), 45523 (R&D Systems, Cat. MAB181), or 45531 (R&D Systems, Cat. MAB182) CCR5 primary antibodies in PBS with 1% FBSΔ for 20 min at RT, followed by 1:500 anti-mouse AlexaFluor 647 (abcam, Cat. ab150107) under the same conditions.

Techniques: Labeling, Software

Journal: Journal of Translational Medicine

Article Title: Identifying CCR5 coreceptor populations permissive for HIV-1 entry and productive infection: implications for in vivo studies

doi: 10.1186/s12967-022-03243-8

Figure Lengend Snippet: CCR5 conformation frequency visualized in JC53 cells. Quantified CCR5 subpopulation frequency in JC53 HeLa derivatives transfected with CCR5-GFP fused to the CT, showing total versus surface CCR5 events. Cells were fixed and labeled with an NT or ECL2 epitope-specific AlexaFluor 647-conjugated mAb ( A ). Cells labeled for total CCR5 events were fixed and permeabilized prior to staining ( B ). Cells were imaged using a Zeiss LSM 800 microscope and ZEN Blue 2.3 software. CCR5-GFP (green); mAb (red); co-localized signals (yellow). Bar size—5 µm

Article Snippet: Each group was stained with one of 1:10 T 21/8 (Invitrogen, Cat. 14-1957-82), CTC5 (R&D Systems, Cat. MAB1802), CTC8 (R&D Systems, Cat. MAB1801), 2D7 (BD Biosciences, Cat. 555991), 45523 (R&D Systems, Cat. MAB181), or 45531 (R&D Systems, Cat. MAB182) CCR5 primary antibodies in PBS with 1% FBSΔ for 20 min at RT, followed by 1:500 anti-mouse AlexaFluor 647 (abcam, Cat. ab150107) under the same conditions.

Techniques: Transfection, Labeling, Staining, Microscopy, Software

Journal: Journal of Translational Medicine

Article Title: Identifying CCR5 coreceptor populations permissive for HIV-1 entry and productive infection: implications for in vivo studies

doi: 10.1186/s12967-022-03243-8

Figure Lengend Snippet: CCR5 conformation frequencies in different cell lines. JC53 ( A ) and U87.CD4 ( B ) cell lines were transfected with CCR5-GFP, grown in culture for 24 h post-transfection, and stained with one of six antibodies: CTC5 (NT), CTC8 (NT), T 21/8 (NT), 2D7 (ECL2), 45531 (ECL2), or 45523 (ECL1/ECL2). All primary antibodies were labeled with a secondary antibody conjugated with AlexaFluor 647. Cells were imaged and colocalization coefficients normalized to total GFP were calculated using ZEN Blue 2.3 software at ×1.3 zoom using 10 different cells. Data was graphed using GraphPad Prism 9. **p < 0.01; ***p < 0.001

Article Snippet: Each group was stained with one of 1:10 T 21/8 (Invitrogen, Cat. 14-1957-82), CTC5 (R&D Systems, Cat. MAB1802), CTC8 (R&D Systems, Cat. MAB1801), 2D7 (BD Biosciences, Cat. 555991), 45523 (R&D Systems, Cat. MAB181), or 45531 (R&D Systems, Cat. MAB182) CCR5 primary antibodies in PBS with 1% FBSΔ for 20 min at RT, followed by 1:500 anti-mouse AlexaFluor 647 (abcam, Cat. ab150107) under the same conditions.

Techniques: Transfection, Staining, Labeling, Software

Journal: Journal of Translational Medicine

Article Title: Identifying CCR5 coreceptor populations permissive for HIV-1 entry and productive infection: implications for in vivo studies

doi: 10.1186/s12967-022-03243-8

Figure Lengend Snippet: Endocytosis and recycling rates of CCR5 surface populations in U87.CD4.CCR5 cells: temperature versus RANTES trigger. U87.CD4 cells were transfected with CCR5-GFP. 24 h post-transfection, cells were divided into groups and treated with or without 100 nM RANTES at 37 °C ( A ). After 0, 15, 30, 45, 60, 75, or 90 min, cells were fixed and then stained with CTC8 (blue lines), 2D7 (green lines), or 45523 (red lines). Colocalization percentages first were normalized to the 0-min timepoint and graphed. To determine recovery rates, U87.CD4 cells were transfected with CCR5-GFP. 24 h post-transfection, cells were divided into groups and treated with RANTES for 90 min at 37 °C. RANTES was removed, and cells were treated with 1 µM cytochalasin D, 0.45 M sucrose, or left as a volume equivalent control for 0, 30, 60, or 120 min at 37 °C ( B ). After treatment, cells were fixed and stained with CTC8 (blue lines), 2D7 (green lines), or 45523 (red lines). Colocalization coefficients were calculated using ZEN Blue 2.3 software at ×1.3 zoom using 10 different cells and graphed as a percentage of the control (without treatment)

Article Snippet: Each group was stained with one of 1:10 T 21/8 (Invitrogen, Cat. 14-1957-82), CTC5 (R&D Systems, Cat. MAB1802), CTC8 (R&D Systems, Cat. MAB1801), 2D7 (BD Biosciences, Cat. 555991), 45523 (R&D Systems, Cat. MAB181), or 45531 (R&D Systems, Cat. MAB182) CCR5 primary antibodies in PBS with 1% FBSΔ for 20 min at RT, followed by 1:500 anti-mouse AlexaFluor 647 (abcam, Cat. ab150107) under the same conditions.

Techniques: Transfection, Staining, Control, Software

Journal: Journal of Translational Medicine

Article Title: Identifying CCR5 coreceptor populations permissive for HIV-1 entry and productive infection: implications for in vivo studies

doi: 10.1186/s12967-022-03243-8

Figure Lengend Snippet: HIV-1 induction of CCR5 internalization. Evaluation of CCR5 subpopulations in non-infected versus HIV-1 infected primary cells. PBMCs isolated from whole blood were grown for 3 days in culture media containing IL-2 and PHA. Cells were cultured for 1 week ( A ) or infected for 3 h with HIV BaL and cultured for 1 week post-infection ( B ) before being stained with one of six antibodies: CTC5 (NT), CTC8 (NT), T 21/8 (NT), 2D7 (ECL2), 45523 (ECL1/ECL2), or 45531 (ECL2). All primary antibodies were labeled with a secondary AlexaFluor 647-conjugated antibody. Cells were then permeabilized and labeled with an anti-CCR5 CT antibody, followed by a secondary DyLight 488-conjugated antibody. Cells were imaged and colocalization coefficients normalized to total DyLight (488 nm) and calculated using ZEN Blue 2.3 software at ×1.3 zoom using 10 different cells. This was repeated in a second donor for validation. Data was graphed using GraphPad Prism 9. *p < 0.05

Article Snippet: Each group was stained with one of 1:10 T 21/8 (Invitrogen, Cat. 14-1957-82), CTC5 (R&D Systems, Cat. MAB1802), CTC8 (R&D Systems, Cat. MAB1801), 2D7 (BD Biosciences, Cat. 555991), 45523 (R&D Systems, Cat. MAB181), or 45531 (R&D Systems, Cat. MAB182) CCR5 primary antibodies in PBS with 1% FBSΔ for 20 min at RT, followed by 1:500 anti-mouse AlexaFluor 647 (abcam, Cat. ab150107) under the same conditions.

Techniques: Infection, Isolation, Cell Culture, Staining, Labeling, Software, Biomarker Discovery