Ccr5 pe clone 3a9

Manufactured by BD

Sourced in United States

CCR5-PE (clone 3A9) is a flow cytometry reagent that binds to the CCR5 receptor on the surface of cells. It is a monoclonal antibody conjugated to the PE (phycoerythrin) fluorescent dye. This reagent can be used to identify and quantify CCR5-expressing cells in various sample types.

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Lab products found in correlation

Ki 67 fitc clone b56, by BD (3 mentions) Cd95 pe cy5 clone dx2, by BD (2 mentions) Cd3 apc cy7 clone sp34 2, by BD (2 mentions) Cd4 pacific blue clone okt4, by BioLegend (2 mentions) Cd8 pe cy7 clone sk1, by BD (2 mentions) Cd28 ecd clone cd28 2, by Beckman Coulter (2 mentions) Ficoll paque plus, by GE Healthcare (2 mentions) Click it edu flow cytometry assay kit, by Thermo Fisher Scientific (1 mentions) Cxcr4 pe clone 12g5, by BD (1 mentions) Cd69 pe clone fn50, by BD (1 mentions) Facscalibur flow cytometer, by BD (1 mentions) Cd3 v500 clone sp34 2, by BD (1 mentions) Cd4 percp cy5.5 clone l200, by BD (1 mentions) Methanol free formaldehyde, by Polysciences (1 mentions) Cyan adp, by Beckman Coulter (1 mentions) Ficoll paque plus, by Cytiva (1 mentions) Live dead fixable aqua, by Thermo Fisher Scientific (1 mentions) Helios fitc clone 22f6, by BioLegend (1 mentions) Cd3 alexa700 clone sp34 2, by BD (1 mentions) Facsdiva software, by BD (1 mentions)

6 protocols using ccr5 pe clone 3a9

Multiparameter Immune Cell Profiling

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Cells were stained using fluorescently conjugated monoclonal antibodies to CD3-PerCP (clone SP34–2), CD4-FITC (clone RPA-T4), in combination with either CCR5-PE (clone 3A9), CXCR4-PE (clone 12G5), or the activation marker CD69-PE (clone FN50), all from BD Biosciences (Franklin Lakes, New Jersey, USA). Corresponding fluorescent isotype controls were used at the same concentrations as the reference antibody. Cells were stained with antibodies by incubation for 30 min at 4°C, washed in PBS-1% FCS and fixed in 1.5% paraformaldehyde. Proliferation assays were performed using the Click-iT EdU Flow Cytometry Assay kit (Life Technologies), as per manufacturer instructions. A gate (PBMC gate) was defined in the analysis to exclude nonviable cells and debris. The percentage of live/dead cells in the PBMC gate and in the CD4⁺ cell population was analyzed using the Live/dead Fixable dead cell stain kit, as described above. Acquisition was performed with a FACScalibur flow cytometer (Becton Dickinson) and CELLQuestPro Software was used for analysis. The cell surface expression levels in the flow cytometry profiles are expressed as mean fluorescence intensity (MFI) indices. The percentage of stained cells is also presented.

Camus C., Matusali G., Bourry O., Mahe D., Aubry F., Bujan L., Pasquier C., Massip P., Ravel C., Zirafi O., Munch J., Roan N.R., Pineau C, & Dejucq-Rainsford N. (2016). Comparison of the effect of semen from HIV-infected and uninfected men on CD4+ T-cell infection. AIDS (London, England), 30(8), 1197-1208.

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PBMC Phenotyping by Flow Cytometry

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PBMC were stained with various combinations of the following monoclonal antibodies: CD3-V500 (clone SP34.2, BD-Biosciences), CD4-PerCp-Cy5.5 (clone L200, BD-Biosciences) or CD4-APC, clone 13B8.2, Beckman-Coulter), CD8-FITC (clone 3B5, Invitrogen, ThermoFisher), CCR5-PE (clone 3A9, BD-Biosciences). After 30 min of incubation at 4°C, cells were washed with cold PBS then fixed in PBS containing 1.6% methanol-free formaldehyde (Polysciences). Data was collected on a three-laser CyAn ADP (Beckman-Coulter) and analyzed on FlowJo version 10 software.

Obregon-Perko V., Hodara V.L., Parodi L.M, & Giavedoni L.D. (2018). Baboon CD8 T cells suppress SIVmac infection in CD4 T cells through contact-dependent production of MIP-1α, MIP-1β, and RANTES. Cytokine, 111, 408-419.

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Comprehensive PBMC Isolation and Immunophenotyping

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PBMC were isolated from whole EDTA blood by Ficoll-Paque Plus (GE Healthcare) gradient centrifugation. Mononuclear cell preparations of intestinal biopsy and necropsy tissues were obtained by mechanical disruption and enzymatic dissociation followed by lymphocyte separation by centrifugation through Ficoll-Paque Plus (Amersham BioScience), as described previously^{54 (link)}. Freshly isolated cells were immunophenotyped using the following antibody panel: CD4 Pacific Blue (clone OKT4; BioLegend; 1:50), CCR5 PE (clone 3A9; BD Biosciences; 1:20), CD28 ECD (clone CD28.2; Beckman Coulter, 1:20), CD95 PE-Cy5 (clone DX2; BD Biosciences; 1:10), CD8 PE-Cy7 (clone SK1: BD Biosciences; 1:100), CD38 APC (clone OK10; NIH Nonhuman Primate Reagent Resource; 1:25), CD3 APC-Cy7 (clone SP34-2; BD Biosciences; 1:100) and Ki67 FITC (clone B56; BD Biosciences; 1:20). Approximately 100,000 CD3+ T cells were acquired for each sample and data analysis was performed using FCS Express software. All dilutions are from the manufacturer’s stock and based on 100 μl total staining volume.

Hao X.P., Lucero C.M., Turkbey B., Bernardo M.L., Morcock D.R., Deleage C., Trubey C.M., Smedley J., Klatt N.R., Giavedoni L.D., Kristoff J., Xu A., Del Prete G.Q., Keele B.F., Rao S.S., Alvord W.G., Choyke P.L., Lifson J.D., Brenchley J.M., Apetrei C., Pandrea I, & Estes J.D. (2015). Experimental colitis in SIV-uninfected rhesus macaques recapitulates important features of pathogenic SIV infection. Nature Communications, 6, 8020.

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Immunophenotyping of Peripheral Blood and Tissue Lymphocytes

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Peripheral blood mononuclear cells were isolated from whole EDTA blood by Ficoll-Paque Plus (GE Healthcare) gradient centrifugation. Mononuclear cell preparations of intestinal biopsy and necropsy tissues were obtained by mechanical disruption and enzymatic dissociation followed by lymphocyte separation by centrifugation through Ficoll-Paque Plus (GE Healthcare), as described previously54 (link). Freshly isolated cells were immunophenotyped using the following antibody panel: CD4 Pacific Blue (clone OKT4; BioLegend; 1:50); CCR5 PE (clone 3A9; BD Biosciences; 1:20); CD28 ECD (clone CD28.2; Beckman Coulter, 1:20); CD95 PE-Cy5 (clone DX2; BD Biosciences; 1:10); CD8 PE-Cy7 (clone SK1: BD Biosciences; 1:100); CD38 APC (clone OK10; NIH Nonhuman Primate Reagent Resource; 1:25); CD3 APC-Cy7 (clone SP34-2; BD Biosciences; 1:100); and Ki67 FITC (clone B56; BD Biosciences; 1:20). Approximately 100,000 CD3+ T cells were acquired for each sample and data analysis was performed using FCS Express software. All dilutions are from the manufacturer's stock and based on 100 μl total staining volume.

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Multi-color Flow Cytometry Analysis of RM

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Multi-color flow cytometric analysis was performed on mononuclear cells isolated from blood and lymph nodes according to standard procedures using monoclonal antibodies directed against RM markers and human markers that also cross-react with the same markers in RM. For optimum staining of intra-cellular markers, permeabilization of cells using the eBioscience FoxP3-permeabalization buffer was performed as recommended by the manufacturers. Pre-determined optimal concentrations of the following antibodies and reagents were used: CD3-Alexa700 (clone SP34-2), CD4-Allophycocyanin-Cy7 (clone OKT-4), Bcl-6-PeTexasRed (clone K112-91), Ki67- FITC (clone B56), CCR5-PE (clone 3A9), CTLA4- BV421 (clone BNI3) from BD, CXCR5-PerCP eFlour 710 (clone MU5BEE), PD1-PeCy7 (clone J105), CD127-PeCy5 (clone eBio-RDR5) from eBioscience and CD20-BV650 or PE-CF594 (clone 2H7), CD25-BV711 (clone BC96), Helios-FITC (clone 22F6) and FoxP3-Allophycocyanin (clone 150D) from Biolegend, and Live/Dead Fixable Aqua from Invitrogen. Flow cytometric data were acquired using LSRII flow cytometer using BD’s FACS DiVA software. Acquired data were analyzed using Flow Jo version9.3.2 following the gating strategy described in Figure1. Further analyses were performed using PRISM (GraphPad) and Excel (Microsoft Office 2011) software.

Chowdhury A., Estrada Del Rio P.M., Tharp G.K., Trible R.P., Amara R.R., Chahroudi A., Reyes-Teran G., Bosinger S.E, & Silvestri G. (2015). Decreased TFR/TFH ratio in SIV-infected rhesus macaques may contribute to accumulation of TFH cells in chronic infection. Journal of immunology (Baltimore, Md. : 1950), 195(7), 3237-3247.

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Multiparametric Caspase Activity Assay

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Peptides (Proteogenix) were received as dry powder and reconstituted with water for use. Peptide (clone G46-6, Biolegend), CCR5-PE (clone 3A9, BD), p24-FITC (clone KC57, Coulter). AAC-11 expression was analyzed by intracellular flow cytometry staining using BD Cytofix/Cytoperm TM buffer set (BD) and anti-AAC-11 primary antibody (ab65836, abcam) at 1/100 dilution followed by a secondary antibody conjugated to A674 (ThermoFisher, A21244) at a 1/1000 dilution. Caspase activity was assayed with the following probes according to the manufacturer's instructions: caspase-1 660-YVAD-FMK probe (FLICA 660 Caspase-1 Assay Kit, ImmunoChemistry Technologies LLC ), caspase-2 FAM-VDVAD-FMK probe (FAM-FLICA Caspase-2 Assay Kit, ImmunoChemistry Technologies LLC ), caspase-3/7 SR-DEVD-FMK probe (SR-FLICA Caspase-3/7 Assay Kit, ImmunoChemistry Technologies LLC ) and caspase-8 FAM-LETD-FMK probe (FAM-FLICA Caspase-8 Assay Kit, ImmunoChemistry Technologies LLC ). FACS sorting was performed on BD Aria and flow cytometry acquisition on BD LSRII.

Mikhailova A., Valle‐Casuso J.C., David A., Monceaux V., Volant S., Passaes C., Elfidha A., Müller‐Trutwin M., Poyet J., & Sáez‐Cirión A. (2020). Anti-apoptotic clone 11 derived peptides induce in vitro death of CD4+ T cells susceptible to HIV-1 infection.

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