Mucosal mononuclear cells were isolated from colonic biopsy specimens by collagenase type II (Sigma, Hamburg, Germany) digestion as described previously2 (link),3 (link). The following antibodies were used for phenotypic analysis: anti-CD3-allophycocyanin (APC), anti-CD3-peridinin chlorophyll protein Cy5.5 (PerCPCy5.5; both clone SP34; BD Biosciences), anti-CD4-PerCPCy5.5, anti-CD4-APC (both L200; BD), anti-CD8-phycoerythrin (PE), anti-CD8-fluorescein (FITC; both DK25; Dako), anti-CD45RA-FITC (ALB11; Beckman Coulter), and anti-CCR5-PE (3A9; BD). Lymphocytes were gated on the basis of characteristic forward and side scatter properties. CD4+ T cells were identified by co-expression of CD3 and lack of CD8 expression and memory CD4+ T cells were identified by lack of CD45RA expression on CD4+ T cells. The total number of analyzed colonic cells was between 360,000 and 550,000 and cells in the CD3+ T cell gate consisted of 14,000 – 44,000 colonic cells. CD4+ T cell counts in fresh whole blood were quantified by the use of Trucount Tubes (BD) according to the manufacturer’s instructions. Data were acquired on the FACS Calibur (BD) and analyzed with FlowJo software version 8.8.4. (BD).
Anti ccr5 pe
Manufactured by BD
Sourced in United States
Anti-CCR5-PE is a fluorescently-labeled antibody that binds to the CCR5 chemokine receptor. It is designed for use in flow cytometry applications to detect and analyze cells expressing CCR5.
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Lab products found in correlation
Anti cd4 apc, by BD (2 mentions)
Facscalibur, by BD (2 mentions)
Anti cxcr4 pe, by BD (2 mentions)
Anti cd4 pe, by BD (2 mentions)
Anti cd38 fitc, by STEMCELL (2 mentions)
Anti cd4 percp cy5, by BD (1 mentions)
Trucount tube, by BD (1 mentions)
Collagenase type 2, by Merck Group (1 mentions)
Anti cd45ra fitc, by Beckman Coulter (1 mentions)
Anti cd8 fitc, by BD (1 mentions)
Anti cd44 pe, by BD (1 mentions)
Anti ifn γ bv421, by BD (1 mentions)
Anti il 17a pe, by BD (1 mentions)
Anti cd69 fitc, by BD (1 mentions)
Anti cxcr3 pe, by BD (1 mentions)
Anti cd25 fitc, by BD (1 mentions)
Anti cd62l apc, by BD (1 mentions)
Facs caliber, by BD (1 mentions)
Quantibrite beads, by BD (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
9 protocols using anti ccr5 pe
1
Isolation and Phenotypic Analysis of Colonic Mononuclear Cells
2
Comprehensive Immune Cell Profiling
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Ab-stained cells were analyzed using FACScaliber (BD Biosciences, USA) CellQuest Software. Anti-CD4-PE, anti-CD4-APC, anti-CD8-FITC, anti-CD25-FITC, anti-CD44-PE, anti-CD62L-APC, anti-CD69-FITC, anti-CCR5-PE, anti-CXCR3-PE, and anti-CXCR4-PE, anti-IFNγ-BV421, anti-IL17A-PE, anti-Thy1.2-BV605 were purchased from BD Biosciences.
3
CD4 and CCR5 Expression Analysis
4
Phenotypic Analysis of PBMC and Liver Cells
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PBMCs were isolated from heparinized blood samples by Ficoll-Paque plus (Amersham, Uppsala, Sweden) density gradient. After isolation, cells were washed two times with RPMI-1640 (Gibco, Auckland, N.Z.) and prepared for further study. The surface and intracellular stainings were performed using the following fluorochrome-conjugated antibodies: anti-CD4-PerCP, anti-CD45RA-FITC, anti-CCR4-PE, anti-CCR5-PE, anti-CCR7-PE, anti-Ki67-PE (BD Pharmingen, San Diego, CA), anti-Tim3-PE, anti-TNFR2-PE (R&D Systems, Minneapolis, MN), anti-Helios-PE, anti-FoxP3-APC (eBioscience, San Diego, CA). For intracellular staining, cells were fixed and permeabilized using the Human FoxP3 Buffer Set (eBiosciences, San Diego, CA) according to the manufacturer’s instructions. Isotype-matched control antibodies were used to define the positive staining populations. Stained cells were acquired and analyzed using a FACSAria cytometer (Becton Dickinson, San Jose, CA, USA) with FACSDiVa software (BD Biosciences).
Liver biopsy specimen was obtained from six chronic HBV infected patients and was chopped and incubated with collagenase-D (0.1%) (Gibco, Waltham, USA) in RPMI-1640 with 10% fetal bovine serum (Gibco, Grand Island, NY). After incubate and filtered through nylon mesh, cells were suspended in RPMI-1640 and then stained with anti-CD4-PerCP, anti-CD45RA-FITC, anti-Tim3-PE and anti-FoxP3-APC.
Liver biopsy specimen was obtained from six chronic HBV infected patients and was chopped and incubated with collagenase-D (0.1%) (Gibco, Waltham, USA) in RPMI-1640 with 10% fetal bovine serum (Gibco, Grand Island, NY). After incubate and filtered through nylon mesh, cells were suspended in RPMI-1640 and then stained with anti-CD4-PerCP, anti-CD45RA-FITC, anti-Tim3-PE and anti-FoxP3-APC.
5
Immunophenotyping of T-cell Subsets
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Freshly isolated PBMCs were immunophenotyped for Treg number, FoxP3 expression (MFI) and CCR5/CxCR4 expression using fluorochrome-conjugated monoclonal antibodies (mAb): anti-CD4 PE/FITC, anti-CD25 PE-Cy7, anti-CCR5 PE, and anti-CxCR4 PE (BD Bioscience, San Jose, CA) for cell-surface markers in combination with intracellular Fork-head box protein 3 (FoxP3) mAb conjugated with Alexa488 (eBioscience, San Diego, CA). Samples were acquired into a flow cytometer (FACS Calibur, BD, USA) and analyzed using Cell-Quest (BD Bioscience)/FlowJo (Treestar) softwares. For further analysis, patients were categorized on the basis of level of surface CD25 expression on CD4+ T-cells viz: CD4+CD25high (top 2% with high CD25 expression), CD4+CD25intermediate (middle 15% with intermediate CD25 expression) and CD4+CD25low/negative (lower 83% with very low or no expression of CD25) cells (Figure 1A & 1B
6
Comprehensive Multiparametric Flow Cytometry Analysis of CD4+ T Cell Subsets and Activation States
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For CD4 T cell subsets sort, PBMC were stained with fluorochrome-labelled mAbs anti-CD28-CY5PE, anti-CCR5-PE, anti-CD3-CY7APC, anti-CD4-BV605, anti-CD8-Pacific blue (BD Biosciences) and anti-CD95-BV785 (in house conjugated, BD Biosciences). LN cells were stained with anti-CD28-CY5PE, anti-CD3-CY7APC, anti-CD4-BV605 (BD Biosciences), anti-CD8-BV570, anti-CXCR5-PE (eBioscience) and anti-CD95-BV785 (in house conjugated, BD Biosciences). PBMC and LN CD4 subsets of interest were sorted and lyzed in proteinase K (100ug mL-1, Sigma Aldrich) for SIV gag qPCR. For assessment of T cell activation and exhaustion, cryopreserved PBMC were thawed and stained with fluorochrome-labelled mAbs anti-CD38-FITC (Stem Cell), anti-Ki67-CY7PE, anti-CD28-CY5PE, anti-CD3-CY7APC (BD Biosciences), anti-HLA-DR-TRPE (Life Technologies), anti-PD-1-BV711, anti-CD95-BV785 (Biolegend), anti-TIGIT-APC (ThermoFisher) and anti-LAG3-PE (R&D). All samples were stained with Aqua LIVE/DEAD Fixable Dead Cell Stain.
7
Phenotypic Analysis of Dendritic Cells and Macrophages
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Analysis of cell-surface phenotype of DCs and MØs was performed by flow cytometry. Cells were labeled with anti-CD4-FITC, anti-CD69-PC5, anti-CD80-FITC, anti-CD1a-PE, anti-HLA-DR-ECD, anti-CD14-PC7, anti-CD86-PC5.5 (all from Beckman Coulter Inc., Brea, CA, USA), anti-CD8-Pacific Blue, anti-CD36-APC, anti-CD197-APC-Cy7 (CCR7) (all from BioLegend, San Diego, CA, USA), anti-CCR5-PE, anti-CD209-PE, anti-CD83-APC (all from BD Biosciences, Franklin Lake, NJ, USA), anti-CXCR4-APC (RandD systems, Minneapolis, MN, USA) and anti-CD163-FITC (MBL International Corp., Woburn, WA, USA). Levels of surface expression on cells were estimated by flow cytometry (Gallios; Beckman) and analyzed using Kaluza software (Beckman).
8
Cytometric Identification of Immune Cells
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Cells from blood, peritoneal lavage and bone marrow were stained with the following antibodies: anti-Gr-1 PerCP, anti-CCR5 PE (BD Biosciences, San Jose, Calif), anti-Annexin V FITC (Southern Biotech, Birmingham, Ala), anti-Ly6G FITC or APC, anti-Ly6C Percp and anti-CD11b APC-Cy7 (BD Biosciences, San Jose, Calif). FACS was carried out in FACSCanto cytometer (BD Biosciences, San Jose, Calif) and analyzed by FCS Express V3 (De Novo Software). For cell sorting, blood was collected from mice 6 h after sepsis and CCR5-positive and negative cells were sorted by FACSAria III cytometer (BD Biosciences, San Jose, Calif) and stained by the May-Grünwald-Giemsa method for morphology analysis. Cells were also collected from the bone marrow of naive mice and inflammatory monocytes (Ly6G -CD11b + Ly6C high ) were sorted.
9
Phenotypic Analysis of T-cells by Flow Cytometry
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Flow cytometry analysis of T-cell phenotype was performed according to the protocols as previously described. [21] [22] [23] Monoclonal antibodies used in this study including anti-CD3-BV605, anti-CD45-PE, anti-CD4-BV421, anti-CD8-APC-R700 anti-CD95-DX2, anti-CD28-CD28.2, anti-HLA-DR-PE-Cy7, anti-CD69-APC and anti-CCR5-PE, were purchased from BD Biosciences (San Jose, CA, USA). Anti-CD38-FITC was obtained from StemCell Technologies (Vancouver, BC, Canada). Anti-CD25-APC was from Biolegend (San Diego, CA, USA), and anti-CD127-PE was a product of Invitrogen (Carlsbad, CA, USA). CD4 + T cell differentiation was identi ed in terms of CD28 and CD95 expression, as CD28 + CD95 + CD4 + T cell de ned as central memory CD4 + T cell (CD4 + Tcm) and CD28 + CD95 -CD4 + T cell as effector memory CD4+ T cell (CD4 + T EM ). CD28 + CD95 + CD8 + T cell was de ned as CD8 + T CM cell whereas CD28 + CD95 -CD8 + T cell was CD8 + T EM cell. In addition, expression of CD25 and CD127 were measured to evaluate regulatory T Cells (Tregs). Activation markers HLA-DR, CD38 and CD69 were measured on CD4 + and CD8 + T cells, and CD4 + CCR5 + T cells were de ned as SIV-infected cells. All dates were acquired and analyzed on a ow cytometer (FACSCanto; Becton Dickinson).
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