ccr5 primary antibodies  (R&D Systems)

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

Journal: BioMed Research International

Article Title: Calcium Dyshomeostasis Alters CCL5 Signaling in Differentiated PC12 Cells

doi: 10.1155/2019/9616248

Figure Lengend Snippet: Western blot analysis of CCRs protein in PC12 cell lines. (a) Approximately 40–60 μ g of total protein was resolved on a 10% SDS-PAGE gel and electroblotted onto nitrocellulose membranes. Membranes were probed with polyclonal anti-CCR1, polyclonal anti-CCR3, or monoclonal anti-CCR5. Representative blots are shown and arrows indicate the main band of the receptors with apparent MW 41 kDa for CCR1, 40 kDa for CCR3, and 46 kDa for CCR5. (b) The bands intensity was densitometrically analyzed and the results are expressed as % of the control PC12 cells obtained after normalization to endogenous β -actin level (± SD). ∗ P < 0.05 (n=7), C: control line, _2: PMCA2-reduced line, _3: PMCA3-reduced line.

Article Snippet: Subsequently, cells were washed once and further incubated for 1 h with the blocking buffer (10% normal goat serum, 0.1% Triton X-100, PBS pH = 7.4), following 1 h incubation with primary antibody against CCR5 diluted in the blocking buffer (1:100, Santa Cruz Biotechnology).

Techniques: Western Blot, SDS Page, Control

Journal: BioMed Research International

Article Title: Calcium Dyshomeostasis Alters CCL5 Signaling in Differentiated PC12 Cells

doi: 10.1155/2019/9616248

Figure Lengend Snippet: Localization of CCR5 in PC12 cell lines. PC12 cells differentiated for 48 h with db-cAMP were fixed and immunostained with antibodies against CCR5 receptor (green) and Na + /K + ATPase (plasma membrane marker, red). Nuclei were stained with Hoechst 33342 (blue). Representative confocal images are presented. Values shown in merged images represent average fluorescence intensity ± SEM (n = 5) of pixels positive in green channel (CCR5) that colocalize with red channel positive pixels (Na + /K + ATPase). ∗ P< 0.05, ∗∗ P < 0.01. Scale bars: 10 μ m. C: control line, _2: PMCA2-reduced line, _3: PMCA3-reduced line.

Article Snippet: Subsequently, cells were washed once and further incubated for 1 h with the blocking buffer (10% normal goat serum, 0.1% Triton X-100, PBS pH = 7.4), following 1 h incubation with primary antibody against CCR5 diluted in the blocking buffer (1:100, Santa Cruz Biotechnology).

Techniques: Clinical Proteomics, Membrane, Marker, Staining, Fluorescence, Control

Journal: BioMed Research International

Article Title: Calcium Dyshomeostasis Alters CCL5 Signaling in Differentiated PC12 Cells

doi: 10.1155/2019/9616248

Figure Lengend Snippet: Analysis of CCL5 effect on calcium transients in PC12 cell lines. Ca 2+ transients were measured in parallel wells with (red lines) or without (black lines) presence of specific inhibitors. (a) SERCA inhibitor, 1 μ M thapsigargin, was added after 50 s (red arrow), and next 50 ng/ml CCL5 was applied after 150 s (black arrow). (b) CCRs inhibitors, 1 nM BX513 for CCR1, 1 μ M SB328437 for CCR3, and 1 nM DAPTA for CCR5, were included just before measurements. 50 ng/ml CCL5 was applied after 150 s (black arrow). All measurements were done in duplicate and the presented traces are average from 5 independent cell cultures (n=10). C: control line, _2: PMCA2-reduced line, _3: PMCA3-reduced line.

Article Snippet: Subsequently, cells were washed once and further incubated for 1 h with the blocking buffer (10% normal goat serum, 0.1% Triton X-100, PBS pH = 7.4), following 1 h incubation with primary antibody against CCR5 diluted in the blocking buffer (1:100, Santa Cruz Biotechnology).

Techniques: Control

Journal: BioMed Research International

Article Title: Calcium Dyshomeostasis Alters CCL5 Signaling in Differentiated PC12 Cells

doi: 10.1155/2019/9616248

Figure Lengend Snippet: Schematic presentation of CCL5 effect on PC12 cells. Downregulation of neurospecific PMCA2 or PMCA3 isoforms in differentiated PC12 cells generated two types of cell response: similar for both lines or characteristic for only one line. The common changes were increased cytosolic Ca 2+ and, as a compensatory mechanism, upregulation of PMCA1 isoform, enlarged expression of SERCA2 and SERCA3, and diminished calmodulin amount [ , ]. The levels of CCR5 and IP 3 R-3 proteins also increased, but the expression of IP 3 R-1 and IP 3 R-2 was lowered (present study). Interestingly, altered IP 3 R isoform composition did not change total IP 3 R protein in the _2 line, while it increased in the _3 line. Also in _3 cells, the amount of PMCA4 increased . These subtle differences could have profound consequences after CCL5/CCR5 activation, since potency to restore the basal Ca 2+ level in the _3 line appears to be higher than in the _2 line, which may be essential for the survival of the cell. Under prolonged Ca 2+ signal in the _2 line due to reduction of the fastest isoform - PMCA2, the subsequent Ca 2+ -mediated processes could increase vulnerability to cell death. Abbreviations used: CaM, calmodulin; CCL5, chemokine C-C motif ligand 5; CCR5, receptor for CCL5; inositol 1,4,5-triphosphate (IP 3 ); IP 3 R, IP 3 receptor; PMCA, plasma membrane Ca 2+ -ATPase; SERCA, sarco/endoplasmic Ca 2+ -ATPase.

Article Snippet: Subsequently, cells were washed once and further incubated for 1 h with the blocking buffer (10% normal goat serum, 0.1% Triton X-100, PBS pH = 7.4), following 1 h incubation with primary antibody against CCR5 diluted in the blocking buffer (1:100, Santa Cruz Biotechnology).

Techniques: Generated, Expressing, Activation Assay, Clinical Proteomics, Membrane