Cervical cells were obtained from Digene cytobrushes, processed within 4 hours, and stained immediately [11 (link)]. The antibodies included: APC-H7-CD3, BV605-CCR6, and APC-CCR5 (all from BD Biosciences, San Jose, CA); BV711-CD8, PE-CCR10, and Alexa-700-human leukocyte antigen (HLA) DR isotope (Biolegend, San Diego, CA); PE-Cy5.5-CD4 (Invitrogen, Carlsbad, CA); and PE-Cy7-cluster of differentiation 38 (CD38) (eBioscience, San Diego, CA). Staining for LIVE-DEAD cells, Pac-blue-CD14, and Pac-blue-CD19 was performed to exclude dead cells, monocytes, and B-cells, respectively (all from Invitrogen). Cells were acquired using the FORTESSA (BD Immunocytometry Systems, San Jose, CA). FlowJo v9.9.3 (FlowJo, LLC, Ashland, OR) was used for the data analysis. The gating strategy is shown in Supplementary Figure 1 .
Pe ccr10
Manufactured by BioLegend
PE-CCR10 is a fluorescently-labeled antibody that binds to the CCR10 chemokine receptor. CCR10 is a G protein-coupled receptor that plays a role in the migration and homing of lymphocytes. The PE (Phycoerythrin) fluorescent dye is conjugated to the antibody, allowing for detection and analysis of CCR10-expressing cells using flow cytometry.
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Lab products found in correlation
Cd3 apc h7, by BD (2 mentions)
Ccr5 apc, by BD (2 mentions)
Pe cy5.5 cd4, by Thermo Fisher Scientific (2 mentions)
Bv605 ccr6, by BD (2 mentions)
Cd8 bv711, by BioLegend (2 mentions)
Alexa 700 human leukocyte antigen dr isotope, by BioLegend (2 mentions)
Pe cy7 cluster of differentiation 38 cd38, by Thermo Fisher Scientific (2 mentions)
Fortessa, by BD (1 mentions)
Cxcr5 apc, by BioLegend (1 mentions)
Cd80 pe cy5, by Thermo Fisher Scientific (1 mentions)
Cd19 percp cy5, by Thermo Fisher Scientific (1 mentions)
H3n2 ha, by Sino Biological (1 mentions)
H1n1 ha, by Sino Biological (1 mentions)
H5n1 ha, by Sino Biological (1 mentions)
Cd138 pe, by Thermo Fisher Scientific (1 mentions)
Hla dr pe cy5, by Thermo Fisher Scientific (1 mentions)
Cd20 apc, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Cd62l, by BioLegend (1 mentions)
4 protocols using pe ccr10
1
Cervical Immune Cell Phenotyping
2
Cervical T Cell Immunophenotyping
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Cervical cells were obtained from Digene cytobrushes, processed within 4 h, and stained immediately to measure T cell frequencies and activation by flow cytometry, as previously described (21 (link)). The gating strategy is shown in Fig. S1 in the supplemental material. The following panel of antibodies was included: APC-H7-CD3, BV605-CCR6, and APC-CCR5 (BD Biosciences); BV711-CD8, PE-CCR10, and Alexa-700-human leukocyte antigen (HLA) DR isotope (BioLegend); PE-Cy5.5-CD4 (Invitrogen); and PE-Cy7-cluster of differentiation 38 (CD38) (eBioscience, Inc.). LIVE-DEAD cell, Pacific-blue-CD14, and Pacific-blue-CD19 (Invitrogen) staining were performed to exclude dead cells, monocytes, and B-cells, respectively. Cells were acquired using an LSRFFortessa (BD Immunocytometry Systems). FlowJo v9.9.3 (FlowJo, LLC) was used for the data analysis.
3
Binding Interactions of Influenza Hemagglutinin Subtypes
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BSA and streptavidin were from Sigma-Aldrich. PBS was from Gibco. TT was supplied by the Statens Serum Institut. All recombinant influenza HA subtypes were from Sino Biological: H1N1 HA (A/California/04/2009; H1), H2N2 HA (A/Japan/305/1957; H2), H5N1 HA (A/Vietnam/1194/2004; H5), H3N2 HA (A/Perth/16/2009; H3) and H7N9 HA (A/Shanghai/1/2013; H7). H1N1 HA (A/New Caledonia/20/1999; H1 wt), the HA delta stem mutants H1 I45R/T49R and H1 45glyc, and the human monoclonal antibodies CR6261 and CH65 were provided by D. Lingwood. HIV-1 92BR-gp120 was provided by D.R. Burton (Scripps Institute, San Diego, CA). Human CpG ODN 2006 was from InvivoGen. IL-15 and IL-6 were from PeproTech. Antibodies used were as follows: goat anti–human IgG (FcΥ specific; Jackson ImmunoResearch Laboratories, Inc.); anti–human CD27-APC, anti–human CD38-FITC, CD138-PE, CD19-Percp Cy5.5, CD20-APC, CD80-PE-Cy5, HLA-DR-PE-Cy5, and anti–human κ-Ig biotin (eBioscience); anti-human CD86-Bv510, CCR10-PE, CD62L, CXCR4-PE-Cy5, CXCR5-APC, and anti–human λ-Ig biotin (BioLegend); and anti–human CD43-PE and anti–human IgD (Miltenyi Biotec).
4
Comprehensive Immunophenotyping of T Cell Subsets
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Blood was collected and processed within 4 hours maximum. An ammonium chloride-based lysing reagent (BD Pharm Lyse, BD Biosciences) was used for erythrocyte deletion of 1ml of blood. After washing, cells were suspended in PBS and stained with Aqua Dye (Invitrogen) for cell viability. Cells were washed again, suspended in staining buffer and divided in four tubes. The four different panels assessed contained some common and some specific antibodies. Common antibodies were: CD3-eFluor 605, CD4-Alexa700 (eBioscience, San Diego, CA), CCR7-Horizon PE-CF594, CD38-Brillant Violet 421, HLA-DR-PerCP-Cy5.5 and CD11c-PE-Cy7 (BD Biosciences). Specific for each panel were: 1) CCR2-PE, CCR5-APC-Cy7, CXCR6-APC (R&D Systems Inc.) and CXCR3-FITC (BioLegend); 2) CD49d (α4)-FITC, β7-APC, CCR9-PE (BD Biosciences) and CD29 (β1)–APC-Cy7 (BioLegend); 3) CD103-FITC, CD54-APC, CD49a (α1)-PE and CD29–APC-Cy7 (BioLegend); 4) CD18-APC, CLA-FITC (BD Biosciences) and CCR10-PE (BioLegend). Cells were acquired using a BD LSRFortessa SORP flow cytometer (Flow Cytometry Platform, IGTP) and analyzed with FlowJo 9.3.2 software (TreeStar). Gates were drawn based on fluorescence minus one-controls and isotypes, and CD3+ CD4- phenotype was considered CD8+ T cells.
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