Flow cytometric analysis was performed up to 3 times pre-transplant and serially post-transplant to characterize peripheral blood immune cell phenotypes. Total T cells and T cell subsets were quantified by complete blood cell count and flow cytometry. Fresh PBMCs were isolated by Ficoll density gradient centrifugation (BD Biosciences, Franklin Lakes, NJ). PBMCs were stained with the following mAbs: CD3 PacBlue, CD95 V450, CD3 Alexa 700, CD4 PerCP-Cy5.5, CD8 V500, CD28 PE-Cy7, CD25 PE-Cy7, IFNy PE-Cy7, CD28 APC, TNF APC, VLA-4 APC, CD11a PE, CD45RA FITC, CD40 FITC, CCR7 APC, CD20 APC (all BD Biosciences). PBMCs (1.5×106) were incubated with appropriately titered antibodies for 15min at 20°C and washed twice. Samples were acquired immediately on a BD LSR II multicolor flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (Tree Star, San Carlos, CA). For the stimulation assay, 1.5×106 PBMCs were cultured in RPMI 1640 (Corning cellgro, Manassas, VA) supplemented with 10% fetal bovine serum and stimulated with 10µM phorbol 12-myristate 13-acetate (PMA) and 200nM ionomycin (Sigma-Aldrich, St. Louis, MO), with 1ul/ml GolgiPlug protein transport inhibitor for 5 h, +/− IL-15 (10ng/mL). PBMCs were were processed with BD Cytofix/Cytoperm Plus kit (BD 555028) per the manufactures recommendation prior to data acquisition.
Cd25 pe cy7
Manufactured by BD
Sourced in United States, Germany
The CD25-PE-Cy7 is a fluorescent-labeled antibody that binds to the CD25 antigen, also known as the interleukin-2 receptor alpha chain. It is used for the detection and analysis of CD25-expressing cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Cd8 fitc, by BD (8 mentions)
Cd3 percp, by BD (8 mentions)
Cd4 apc h7, by BD (7 mentions)
Cd8 apc h7, by BD (7 mentions)
Cd4 fitc, by BD (7 mentions)
Cd127 bv421, by BD (6 mentions)
Cd45ro apc, by BD (6 mentions)
Cd4 apc cy7, by BD (6 mentions)
Cd56 apc, by BD (5 mentions)
Cd19 pe cy7, by BD (5 mentions)
Foxp3 pe, by BD (5 mentions)
Cd4 pe, by BD (5 mentions)
Facscanto 2, by BD (4 mentions)
Cd25 apc, by BD (4 mentions)
Cd62l fitc, by BD (4 mentions)
Cd3 fitc, by BD (4 mentions)
Cd3 bv421, by BD (4 mentions)
Il 17 apc, by R&D Systems (4 mentions)
Cd11b pe, by BD (4 mentions)
Cd45ra fitc, by BD (3 mentions)
49 protocols using cd25 pe cy7
1
Flow Cytometric Immune Profiling in Transplant
2
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).
3
Profiling Treg and Th17 Cells in PBMCs
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To test CD4+CD25hiFoxp3+Treg cells, 1 × 106 PBMCs were stained with CD4 FITC (eBioscience, San Diego, California, USA, Cat# 11-0048-42) and CD25 PE-CY7 (BD Bioscience, Cat# 506225). To detect Th17 cells, 1 × 106 PBMCs were adjusted concentration as 5 × 105/ml in RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 200 U/ml penicillin, and 100 μg/ml streptomycin and cultured in 24-well plates overnight. Before stimulation, the supernatants were collected for IL-17 ELISA test. The concentration of PBMCs were stimulated with 50 ng/ml phorbol myristate acetate (PMA, Sigma-Aldrich, St. Louis, Missouri, USA, Cat# P8139) and 500 ng/ml ionomycin (Sigma-Aldrich, Cat# I9657) for 4 hours. End in 2 hours after incubation, 1 ul/ml brefeldin A solution (BFA, Biolegend, Cat# 420602) was added to the culture system. Then PBMCs were stained with CD4 FITC and CD25 PE-CY7. After the fixation and permeabilization step described above, the cells were stained with IL-17A PerCP-Cy5.5 (BD Bioscience, Cat# 560799). All steps are according to the manufacturer's protocol (Supplementary 4 ). Acquisition was performed on a FACS Aria II flow cytometer (BD biosciences, USA) and then analyzed using Flowjo software version 7.6.1 (Tritar Inc., San Carlos, California, USA).
4
Multiparametric Flow Cytometry of tPCLS
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Single‐cell suspensions of tPCLS were generated as described above. Single‐cell suspensions were blocked with FcR blocking reagent (Miltenyi Biotec) in 0.5% PBS‐BSA for 20 min, stained with fluorochrome‐conjugated antibodies, and analyzed on a FACSSymphony A5SE flow cytometer (BD Biosciences). Live single cells were identified by FSC and SSC characteristics. The data were analyzed using FlowJo V10 (TreeStar). All antibodies and secondary reagents were titrated to determine optimal concentrations. Comp‐Beads (BD) were used for single‐color compensation to create multicolor compensation matrices. For gating, fluorescence minus one control was used. The instrument calibration was controlled daily using Cytometer Setup and Tracking Beads (BD Biosciences). The following antibodies were used: CD3‐BUV805 (#612896, BD Biosciences), CD4‐BB630 (#562316, BD Biosciences), CD8‐BV650 (#743067, BD Biosciences), CD14‐PerCP‐Cy5.5 (#561116, BD Biosciences), CD15‐BUV805 (#742057, BD Biosciences), CD16‐BV650 (#563692, BD Biosciences), CD19‐APC‐H7 (#560252, BD Biosciences), CD25‐PE‐Cy7 (#557741, BD Biosciences), CD33‐BV510 (#563257, BD Biosciences), CD45‐AF700 (#368514, BD Biosciences), CD80‐BV711 (#740801, BD Biosciences), CD206‐PE/Cy7 (#321124, BioLegend), CD326‐FITC (#324203, BioLegend), HLA‐DR‐APC/Fire750 (#307658, BioLegend), MerTK‐BV421 (#367603, BioLegend).
5
Comprehensive CD4+ T Cell Profiling
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One day post isolation FACS analysis was used to determine the percentages of various CD4+ T cell subsets, like naïve (TN), effector memory (TEM), central memory (TCM), T helper 17 cells (Th17), follicular helper T cells (Tfh) and regulatory T cells (Treg). For this the following antibodies were used: CD4-APC H7 (BD Biosciences, 560158), CD45RA-PE Cy7 (BD Biosciences, 560675), CD45RO-PerCP Cy5.5 (BD Biosciences, 560607), CCR7-APC (BioLegend, 353214), CCR6-PE (BD Biosciences, 559562), CXCR5-PerCP Cy5.5 (BioLegend, 335001), CD127-PE Cy7 (BD Biosciences, 560822), CD25-Horizon V450 (BD Biosciences, 560355). Further, the expression of different receptors on unstimulated as well as stimulated CD4+ T cells was determined using the following antibodies: CD4-APC H7, TCR-CD3-APC H7 (BD Biosciences, 560275), CD28-PerCP Cy5.5 (BD Biosciences, 560685), MHC-I-Horizon V450 (BD Biosciences, 561346), FAS-PE (BD Biosciences, 556641), FAS-L-PE (BioLegend, 306407), PD1-APC (BD Biosciences, 558694), PD1-L-PE Cy7 (BD Biosciences, 558017), TRAIL-PE (BD Biosciences, 550516), CTLA-4-PE (BD Biosciences, 555853), CD69-Horizon V450 (BD Biosciences, 560740), CD25-PE Cy7 (BD Biosciences, 557741), CXCR4-PerCP Cy5.5 (BD Biosciences, 560670) and CCR5-PE Cy7 (BD Biosciences, 557752). Cells were analyzed using the BD FACS Canto II with FACSDiva software.
6
Immunophenotyping of T Cell Subsets
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Samples were stained without further separation to minimize selective losses shortly after collection. Combinations of the directly conjugated monoclonal antibodies CD3-V500, CD4-PerCP, CD56-PE, CD127-BV421, CD25-PE-CY7, CD25-APC, CD62L-FITC, CD69-APC, CD45RA-FITC, CCR7-PE, Ki67-FITC, Foxp3-APC (BD Bioscience, Mountain View, CA, USA), CD16-APC/CY7, HLADR-APC/CY7 (Biolegend, San Diego, CA, USA) and their relative allotypes were used in individual 8-color flow cytometry assays to analyze the immunophenotype of regulatory T cells and effector T cells.
Intracellular staining was performed using the Intracellular Staining Kit (eBioscience, San Diego, CA, USA). The expression of Ki67 was determined in freshly isolated CD4+CD25highFoxp3+ regulatory T cells. The cellular secretion and function of cytokines were determined after incubation of cells for 5 h with phorbol myristate acetate (PMA) (100 ng/ml) plus ionomycin (2 ug/ml, all reagents from Sigma Chemical Co., St. Louis, MO, USA) to stimulate maximal production of IL-17 and IFN-γ; GolgiStop (0.7 μl/ml) was added to the samples during the last 4 hours to sequester the proteins in the cytoplasm [22 (link),23 (link)].
Intracellular staining was performed using the Intracellular Staining Kit (eBioscience, San Diego, CA, USA). The expression of Ki67 was determined in freshly isolated CD4+CD25highFoxp3+ regulatory T cells. The cellular secretion and function of cytokines were determined after incubation of cells for 5 h with phorbol myristate acetate (PMA) (100 ng/ml) plus ionomycin (2 ug/ml, all reagents from Sigma Chemical Co., St. Louis, MO, USA) to stimulate maximal production of IL-17 and IFN-γ; GolgiStop (0.7 μl/ml) was added to the samples during the last 4 hours to sequester the proteins in the cytoplasm [22 (link),23 (link)].
7
Multiparametric Flow Cytometry Analysis
8
Multiparametric Flow Cytometry Profiling
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Whole blood was drawn in EDTA tubes and the cells were incubated with fluorochrome-conjugated antibodies against human CD3-FITC, HLA-DR-FITC, CD45RA-FITC, CD8-PE, CD16+56-PE, CD69-PE, CD28-PE, CD3-PerCP, CD45-PerCP, CD45RO-PerCP-Cy5.5, CD4-PE-Cy7, CD25-PE-Cy7, CD25-APC, CD39-APC, CD62L-APC, CD8-APC-H7, CD4-APC-H7, CD3-Horizon V450, CD56-Horizon V450, all from BD Biosciences (San Jose, USA). Anti-Foxp3-PE was purchased from eBioscience (San Diego, USA) and Helios-Pacific Blue from Biolegend (San Diego, USA). Lymphocyte subpopulations from peripheral blood were measured by 4- or 7-color combinations. TruCount™ tubes (BD Biosciences) were used for assessment of absolute numbers (cells/μl) of leukocytes (CD45), and major lymphocyte populations; T lymphocyte (CD3), T helper (CD4), T cytotoxic (CD8), B (CD19) and NK (CD16+CD56) cells, as described by the manufacturer. Absolute numbers of subpopulations were derived from the major populations. Proportions of major populations were given as % of lymphocytes and proportions of subpopulations were given as % of their parent population.
9
Isolation and Characterization of Human Regulatory T Cells
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Tregs were isolated with the CD4+CD25+highCD127−/dim Regulatory T Cell Isolation Kit II (human) (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) according to the manufacturer's instructions. In brief, peripheral blood mononuclear cells (PBMCs) were isolated with a Ficoll gradient (Biochrom AG, Berlin, Germany). The CD4+CD25+CD127− T Cell Biotin-Antibody Cocktail II was used to negatively enrich for CD4+CD127− cells followed by a positive selection of CD25+ cells.
To assess the purity of these CD4+CD25+highCD127−/dim cells, samples and PBMCs were tested by flow cytometry. Hence, cells were incubated with the appropriate amount of extracellular antibody and prepared for intracellular staining with FoxP3-Alexa Fluor 647 (BioLegend) using the Human FoxP3 Buffer Set (BD Biosciences). For extracellular staining, CD25-PE-Cy7, CD4-FITC (both BD Biosciences), and CD127-Pacific Blue (BioLegend) were used. In all experiments, the purity of CD4+CD25+highCD127−/dimFoxP3+ cells was ≥95%.
To assess the purity of these CD4+CD25+highCD127−/dim cells, samples and PBMCs were tested by flow cytometry. Hence, cells were incubated with the appropriate amount of extracellular antibody and prepared for intracellular staining with FoxP3-Alexa Fluor 647 (BioLegend) using the Human FoxP3 Buffer Set (BD Biosciences). For extracellular staining, CD25-PE-Cy7, CD4-FITC (both BD Biosciences), and CD127-Pacific Blue (BioLegend) were used. In all experiments, the purity of CD4+CD25+highCD127−/dimFoxP3+ cells was ≥95%.
10
Isolation of Treg and non-Treg Subsets
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For TCR sequencing purposes, CD3+CD4+CD25highCD127low Tregs and CD3+CD4+CD25low/intCD127int/high non-Tregs were isolated from frozen PBMCs and SFMCs, using the FACS Aria III (BD, Franklin Lakes, NJ). Antibodies used for sorting were: anti-human CD3-BV510 (BioLegend, San Diego, CA), CD4-FITC (eBioscience, Frankfurt am Main, Germany), CD25-PE/Cy7 (BD), and CD127-AF647 (BioLegend). To check for FOXP3 expression of the sorted populations anti-human FOXP3-eF450 (eBioscience) was used.
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