Flow cytometry analysis was performed to quantify lymphocyte subsets on BD LSR II flow cytometer with BD FACS DIVA software (BD Biosciences, Heidelberg, Germany). The following antibodies were used for phenotypic analysis: CD45-FITC, CD19/20-PE, CD14-Cy-7, CD25-FITC, CD127-PE, CD45RA-APC, CD3-APC Cy-7, CD8-FITC, CD4-PE, CD4-APC Cy7, CD8-APC Cy7, CD62L-FITC, CCR7-PE, CD27-FITC, CD62L-PE, (Beckton Dickinson, Franklin Lakes, NJ, USA), CD3-ECD, CD56-APC, CD45RO-ECD (Beckman Coulter, Miami, FL, USA).30 (link)
Ccr7 pe
Manufactured by BD
Sourced in United States
CCR7-PE is a fluorescent-labeled antibody that binds to the CCR7 receptor. CCR7 is a chemokine receptor that plays a role in lymphocyte trafficking and homing. The PE (Phycoerythrin) fluorescent label allows for the detection and analysis of CCR7-expressing cells using flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Cd3 fitc, by BD (6 mentions)
Cd8 fitc, by BD (5 mentions)
Facsdiva software, by BD (5 mentions)
Cd45ra pe cy7, by BD (5 mentions)
Cd80 pe, by BD (5 mentions)
Cd45ra apc, by BD (4 mentions)
Cd62l fitc, by BD (4 mentions)
Cd27 fitc, by BD (4 mentions)
Facscanto 2, by BD (4 mentions)
Cd3 percp, by BD (4 mentions)
Cd8 apc h7, by BD (4 mentions)
Cd86 fitc, by BD (4 mentions)
Cd4 apc cy7, by BD (3 mentions)
Cd62l pe, by BD (3 mentions)
Cd8 apc cy7, by BD (3 mentions)
Pd 1 pe, by Thermo Fisher Scientific (3 mentions)
Hla dr fitc, by BD (3 mentions)
Pd 1 bv421, by BD (3 mentions)
Cd4 percp cy5, by BD (3 mentions)
Facscanto, by BD (3 mentions)
33 protocols using ccr7 pe
1
Flow Cytometry Profiling of Lymphocyte Subsets
2
Flow Cytometry Profiling of Lymphocyte Subsets
3
Multicolor Flow Cytometry of Human Lymphocytes
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Freshly obtained human lymphocytes were stained with the following fluorescent antibodies against human leukocyte surface markers: CD4-peridinin-chlorophyll-cyanine 5 (PerCP-Cy5.5), CD25-phycoerythrin (PE) or -allophycocyanin (APC), CD45RA-fluorescein isothiocyanate (FITC), CCR7-PE, CCR5-FITC, CCR4-PE-Cy7, CD62L-PE, Ki67-PE or -APC from (BD Biosciences, USA), and LAP (PE and APC) from (R&D Systems, USA). Intracellular staining with Foxp3-APC or -FITC and CTLA-4-APC antibodies was performed after treatment with fixation and permeabilization buffers (eBiosciences, USA), according to the manufacturer's protocol. Intracellular staining for granzyme B, perforin, and TGF-β was performed after stimulation with PMA and ionomycin for 5 h with Golgi stop. The stimulated cells were first reacted with antibodies against the surface markers CD4 and LAP, then fixed and permeabilized with Cytofix/Cytoperm buffer (BD Biosciences, USA), and finally stained with TGF-ß-PE, granzyme B-FITC, or perforin-PE antibodies. The fluorescence intensity was analyzed using BD FACSCalibur (BD Biosciences, USA) and FlowJo software (Tree Star, USA).
4
Multiparameter Flow Cytometry of Immune Cells
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Single cell suspensions were stained with monoclonal antibodies for 1 hour at 4°C. One to four‐color flow cytometry was performed (FACS LSR, Becton Dickinson). Data were acquired using the CellQuest software (Becton Dickinson) using at least 100 000 events gated for live cells.
The following conjugated rat anti‐mouse mAbs were used: CD45‐PerCP, CD11b‐APC, CCR4‐PE, CCR5‐PE, CCR7‐PE, CCR10‐PE and CXCR4‐PE (BD Biosciences); CD49b‐APC (Biolegend); CD226‐PE, CD4‐FITC, CD25‐APC, CD8a‐FITC, PD1‐PE, CTLA4‐PE, PDL1‐PE‐Cy7, PDL2‐FITC, CD47‐APC (eBiosciences), CD11c‐FITC, GR1‐FITC (Miltenyi Biotech), CD206 (Santa Cruz), and anti‐mouse Foxp3 staining set PE (eBioscience).
The following conjugated rat anti‐mouse mAbs were used: CD45‐PerCP, CD11b‐APC, CCR4‐PE, CCR5‐PE, CCR7‐PE, CCR10‐PE and CXCR4‐PE (BD Biosciences); CD49b‐APC (Biolegend); CD226‐PE, CD4‐FITC, CD25‐APC, CD8a‐FITC, PD1‐PE, CTLA4‐PE, PDL1‐PE‐Cy7, PDL2‐FITC, CD47‐APC (eBiosciences), CD11c‐FITC, GR1‐FITC (Miltenyi Biotech), CD206 (Santa Cruz), and anti‐mouse Foxp3 staining set PE (eBioscience).
5
Isolation and Characterization of T-Cell Subsets
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By use of an AriaII FACS sorter™ (Becton Dickinson, BD, Franklin Lakes, NJ), pure CD28POS cells (purity 98% [95–100%]) were isolated. PBMCs were stained with CD3 Brilliant Violet 510 (BioLegend, San Diego, CA), CD4 Pacific Blue (BD, Franklin Lakes, NJ), CD8 APC-Cy7 (BD Pharm, San Diego, CA), CD28 APC (BD), and the viability dye 7-AAD PerCP (BD). Pure memory subsets (≥95% pure) were isolated using CD3 Brilliant Violet 510 (BioLegend), CD45RO PE-Cy7 (BD) and CCR7 PE (BD): naïve (TN cells: CCR7+CD45RO-), central-memory (TCM cells: CCR7+, CD45RO+), effector-memory (TEM cells: CCR7-, CD45RO+), and end-stage terminally-differentiated EMRA (TEMRA cells: CCR7-CD45RO-) T-cells.
6
Phenotypic Analysis of Lymphocyte Subsets in Ovarian Cancer
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Peripheral lymphocytes (OC patients: n = 27; normal: n = 10) and ascites-associated lymphocyte subsets (n = 43) were characterized using the following antibodies for surface expression by flow cytometry (FACSCanto BD Bioscience): anti-human CD19 FITC, CD45RA FITC, CD8 APC (all Miltenyi Biotec), CD16 PE-Cy7 and CD335 (NKp46) eFluor450 (both eBiosciences), CD4 PE-Cy7 (Southern Biotech), CD3 APC (Biolegend), CCR7 PE (BD Biosciences), and NKG2D FITC (abcam). Corresponding isotype-matched controls were purchased from Miltenyi Biotec, BD Biosciences and eBioscience. TAMs in ascites were analyzed for surface expression of CD14, CD163, CD206 and intracellular expression of CD68 by flow cytometry, as described previously.36 (link)
7
Cryopreserved PBMC Immune Phenotyping
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Cryopreserved PBMCs were used for immune phenotyping. Peripheral blood mononuclear cells were thawed and stained with monoclonal antibodies (mAbs) (30 minutes at 4°C in the dark). The following directly conjugated mAbs were used for cell surface marker staining: CD3 V500, CD4 PE-Cy7, CD8 Pacific Blue, CD45RA PE-Cy7, CCR7 PE, HLA-DR FITC, CD38 PE, CD27 PerCP Cy5.5, CD28 PerCP Cy5.5, CD57 APC (BD Biosiences, San Jose, CA), CD4 APC eFluor780, CD27 APC eFluor780, and PD-1 PE (eBioscience, San Diego, CA). Fluorescence was measured with the FACS Canto II (BD Biosciences). The proportion of T cells expressing each marker and the mean fluorescence intensity were determined using FlowJo 7.6 (TreeStar, Ashland, OR).
8
Comprehensive Immune Cell Profiling
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The monoclonal antibodies used in this study included CD3 PerCP, CD5 APC-R700, CD8-FITC, CD11c PE-Cy5, CD14 APC-H7, CD19 PE-Cy5, CD25 PE-Cy7, CD24 FITC, CD27 PE-Cy7, CD28 PE-Cy5, CD38 APC-H7, CD45 V500, CD45RO APC, CD56 APC, CD57 FITC, CD80 PE, CD86 APC, CD123 APC, CD127 BV421, CCR7 PE, TIGIT BV421, HLA DR V450, TIM3 PE, CXCR5 APC-R700, PD1 BV421, and PDL1 PE-Cy7 (BD Biosciences).
9
Multiparametric Characterization of CD4+ T Cells
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Peripheral blood mononuclear cells (PBMCs) were obtained after Ficoll density centrifugation (Sigma-Aldrich, Burlington, MA). CD4+ T cells were purified from blood using negative selection on magnetic columns (CD4 T-cell isolation kit, human; Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were stained using the following antihuman antibodies: CXCR5-BV605, CD45RA-FITC, CCR7-PE, CXCR3-BV711, CCR6- PercpCy5.5, PD-1-BUV737, CD25-BV786, and CD127-BUV395 (BD Biosciences, Franklin Lakes, NJ). For CSF samples and transmigration assays, CCR7-PE was replaced by a CD4-PE (Beckman Coulter, Brea, CA). Cells were analyzed by flow cytometry (FACS Celesta, BD Biosciences). Dead cells were excluded from the analysis using live/dead Amcyan staining (Invitrogen, Waltham, MA).
10
PBMC Isolation and T-Cell Subpopulation Sorting
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Peripheral blood was collected in heparin-containing vacutainer tubes (Becton Dickinson, Franklin Lakes, USA) and peripheral blood mononuclear cells (PBMC) were freshly isolated by density gradient centrifugation using Lymphoprep (Axis-Shield, Oslo, Norway) according to the manufacturer’s protocol. CD4 T cells were sorted by fluorescence-activated cell sorting (FACS) as CD3+CD4+ and CD8 T cells as CD3+CD4-. Within the CD4 and CD8 T-cell subsets, naïve (CD45RO-) and memory (CD45RO+), truly TNAIVE (CD45RO-CCR7+) and terminally differentiated (TEMRA) cells (CD45RO-CCR7-); as well as CD3+ and CD31- populations were isolated using combinations of the following anti-human monoclonal antibodies: CD3-e450, CD4-A647 (eBioscience, Vienna, Austria), CD45RO-FITC, CCR7-PE, CCR7-PECY7 and CD31-PE (BD Bioscience, Breda, The Netherlands). See Figure A in in S1 File for sorting schemes.
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