Multi-color flow cytometry analysis was performed on PBMCs from all time points by staining for 30 minutes at 4°C with CD3-V450, CD8-FITC or APC, ICOS-PE, HLA-DR-PerCP-Cy5.5, CD25-PE-Cy7, CD45RA-PerCP-Cy5.5, CD62L-FITC, CD127-V450, PD-1-PE, Tim-3-AF700, CD4-APC-Cy7 (BD Biosciences, San Jose, CA), CCR7-PE-Cy7 (R&D Systems, Minneapolis, MN), CTLA-4-FITC (LSBio, Seattle, WA) and FoxP3-APC (eBioscience, San Diego, CA) for T cells. For natural killer (NK) cells, CD3-V450, CD16-APC-Cy7, CD56-PE-Cy7 and Tim-3-AF700 (BD) were used. For myeloid-derived suppressor cells (MDSCs), CD33-PE, CD11b-APC-Cy7, HLA-DR-PerCP-Cy5.5, CD14-V450 and CD15-APC (BD) were used. 1×105 cells were acquired on an LSRII (BD), and data was analyzed using FlowJo software (Tree Star Inc., Ashland, OR). The appropriate isotype controls were used, and dead cells were excluded from the analysis.
Cd45ra percp cy5
Manufactured by BD
Sourced in Germany
CD45RA-PerCP-Cy5.5 is a fluorophore-conjugated antibody that binds to the CD45RA isoform of the CD45 protein. It is designed for use in flow cytometry applications to identify and quantify cell populations expressing the CD45RA antigen.
Automatically generated - may contain errors
Lab products found in correlation
Pd 1 pe, by BD (3 mentions)
Cd8 fitc, by BD (2 mentions)
Cd25 pe cy7, by BD (2 mentions)
Cd11b apc cy7, by BD (2 mentions)
Cd4 apc cy7, by BD (2 mentions)
Cd33 pe, by BD (2 mentions)
Cd3 v450, by BD (2 mentions)
Cd14 v450, by BD (2 mentions)
Cd62l fitc, by BD (2 mentions)
Hla dr percp cy5, by BD (2 mentions)
Cd15 apc, by BD (2 mentions)
Cd127 v450, by BD (2 mentions)
Foxp3 apc, by Thermo Fisher Scientific (1 mentions)
Cd56 pe cy7, by BD (1 mentions)
Cd16 apc cy7, by BD (1 mentions)
Icos pe, by BD (1 mentions)
Tim 3 af700, by BD (1 mentions)
Anti cd3 bv421, by BD (1 mentions)
7 aminoactinomycin d 7 aad, by BioLegend (1 mentions)
Cd56 bv421, by BD (1 mentions)
4 protocols using cd45ra percp cy5
1
Multiparametric Flow Cytometry Analysis
2
Multiparametric Flow Cytometry Phenotyping
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For MDSCs, isolated PBMCs were stained with anti‐CD3‐BV421 (UCHT1/562426), CD19‐BV421 (HIB19/562440), CD56‐BV421 (NCAM16.2/562751), CD20‐BV421 (2H7/562873), CD11b‐BB515 (ICRF44/564517), CD15‐PerCP‐Cy 5.5 (HI98/560828), CD14‐APC (M5E2/555399), and HLA‐DR‐PE (G46‐6/555812) antibodies (BD Biosciences, San Jose, CA, USA) for 45 min. For lectin‐type oxidized LDL receptor 1 (Lox‐1) expression on PMN‐MDSCs, lox‐1‐BV421 (15C4/358610) (Biolegend, San Diego, CA, USA) was used. For Treg cells, isolated PBMCs were stained with anti‐CD4‐FITC (RPA‐T4/555346), CD25‐APC (M‐A251/555434), CD45RA‐PerCP‐Cy 5.5 (HI100/563429), and Foxp3‐PE (259D/C7/560046) antibodies (BD Biosciences) for 45 min. For CD8+ T cells, isolated PBMCs were stained with anti‐CD8‐APC (RPA‐T8/555369) (BD Biosciences), CD39‐PE‐Cy7 (A1/328212) (Biolegend), and PD‐1‐PerCP‐Cy5.5 (EH12.1 /561273) (BD Biosciences) antibodies. Samples were washed twice and then read on a BD FACSVerse (BD Biosciences) flow cytometer. Dead cells were excluded using 7‐Amino‐Actinomycin D (7AAD) (Biolegend). Gating strategies for PMN‐MDSCs, M‐MDSCs, and CD8+ T cells are shown in Supporting information Fig. 1A‐C. All the process of T‐cell assays and flow cytometry analysis (linear axis) adhered to the guidelines of MIATA compliant and Cossarizza [30 ].
3
Immunophenotyping of T-cell Subsets in HIV Infection
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Lymphocyte surface phenotypes were evaluated by flow cytometry on fresh peripheral blood of HIV-infected women alone, using fluorochrome-labeled antibodies: L/D-BV510 (Miltenyi Biotech, Germany); CD4-APC-H7, CD8-PE-Cy7, CD38-PE, CD45R0-PerCPCy5.5, CD45RA-PerCPCy5.5, CD127-APC, CD31-FITC, CCR7-APC, CD103-PE, CD95-FITC, CD69-FITC, PD1-PE (BD Biosciences, Palo Alto, CA).
T-cell subsets were defined as: naive CCR7+ CD45RA+, central memory CCR7+CD45RA−, effector memory CCR7− CD45RA−, and terminally differentiated CCR7− CD45RA+ subsets. Besides, we measured: activation (CD45R0/CD38/CD69), apoptosis (CD95), exhaustion (PD-1), IL7R (CD127), and Recent Thymic Emigrants (CD31 or CD103) on both CD4 and CD8 T-cell subsets.
Briefly, 1 × 106 peripheral blood mononuclear Cells were stained with the appropriate antibodies for 20 minutes at 4°C in the dark, washed and then acquired using FACSVerse cytometer (BD Biosciences, Palo Alto, CA).
T-cell subsets were defined as: naive CCR7+ CD45RA+, central memory CCR7+CD45RA−, effector memory CCR7− CD45RA−, and terminally differentiated CCR7− CD45RA+ subsets. Besides, we measured: activation (CD45R0/CD38/CD69), apoptosis (CD95), exhaustion (PD-1), IL7R (CD127), and Recent Thymic Emigrants (CD31 or CD103) on both CD4 and CD8 T-cell subsets.
Briefly, 1 × 106 peripheral blood mononuclear Cells were stained with the appropriate antibodies for 20 minutes at 4°C in the dark, washed and then acquired using FACSVerse cytometer (BD Biosciences, Palo Alto, CA).
4
Multi-Dimensional Immune Profiling
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