Dual chamber transmission assay was performed as previously described [28 (link)]. HeLa cells were seeded into an upper chamber of a transwell plate (Sigma-Aldrich, Liège, Belgium), while TZM-bl cells were seeded in the lower chamber. Trans Epithelial Electric Resistance (TEER) was measured using Millicell-ERS Volt-Ohm Meter. HIV-1 ADA-M (200 pg) and drugs were added to the upper chamber after 4 days when TEER reached 150 Ohm/cm2. 24 h after infection luciferase value of TZM-bl cells lysate was measured using the Luciferase System Kit and the POLARstar Omega Plate Reader. Data were analyzed using GraphPrism. The HIV-1 DC-SIGN transmission assay was performed as previously described [27 (link)]. To investigate the cellular activation induced by CE or piceatannol, CD25 and CD69 expression was measured on PBMCs after incubation with CE/piceatannol or 10 µg/mL PHA-P for 24 h at 37 °C using FITC-conjugated anti-CD4, PE/Cy7-conjugated anti-CD25, PE-conjugated anti-CD69 mAbs (Biolegend, Amsterdam, Netherlands), and the near-IR fluorescent reactive dye.
Pe cy7 conjugated anti cd25
Manufactured by BioLegend
Sourced in United States
PE-Cy7-conjugated anti-CD25 is a fluorescently labeled antibody that binds to the CD25 cell surface antigen. CD25 is the alpha subunit of the interleukin-2 receptor and is expressed on activated T cells, regulatory T cells, and other immune cell types. The PE-Cy7 fluorescent label allows for the detection and analysis of CD25-expressing cells using flow cytometry.
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Lab products found in correlation
Fitc conjugated anti cd4, by BioLegend (1 mentions)
Transwell plate, by Merck Group (1 mentions)
Apc conjugated anti cd3, by BD (1 mentions)
Fitc conjugated anti cd45ra, by BD (1 mentions)
Apc cy7 conjugated anti cd4, by BD (1 mentions)
Apc conjugated anti hla dr, by BD (1 mentions)
Pe conjugated anti cd38, by BD (1 mentions)
Pe cy7 conjugated anti cd3, by BD (1 mentions)
Apc conjugated anti cd127, by BioLegend (1 mentions)
Apc conjugated anti cd69, by BioLegend (1 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
Apc cy7 conjugated anti il 17a, by BioLegend (1 mentions)
Pe conjugated anti ctla4, by BioLegend (1 mentions)
Clone 150d, by BioLegend (1 mentions)
Alexa fluor 488 conjugated anti foxp3, by BioLegend (1 mentions)
Pe cy7 conjugated anti ccr4, by BioLegend (1 mentions)
Pe conjugated anti ki67, by BioLegend (1 mentions)
Pacific blue conjugated anti, by BioLegend (1 mentions)
Fitc conjugated, by BioLegend (1 mentions)
Lsrfortessa cell analyzer, by BD (1 mentions)
4 protocols using pe cy7 conjugated anti cd25
1
Dual Chamber Transmission Assay for HIV-1
2
Phenotypic Analysis of CD4+ T Cell Subsets
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Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by Ficoll centrifugation. The following monoclonal antibodies (mAbs) and reagents were used in this study: PE-Cy7-conjugated anti-CD3, APC-conjugated anti-CD3, APC-Cy7-conjugated anti-CD4, PE-conjugated anti-CD38, APC-conjugated anti-HLA-DR, APC-Cy7-conjugated anti-HLA-DR, FITC-conjugated anti-CD45RA, PerCP-Cy5.5-conjugated anti-CCR7 (BD Biosciences, USA); Violet-conjugated anti-CD38, FITC-conjugated anti-CD38, PE-Cy7 conjugated anti-CD25, APC conjugated anti-CD69, APC conjugated anti-CD127, Amcyan-conjugated anti-CD45RA (BioLegend, San Diego, CA, USA). For the expression of all markers, flow cytometric gating was defined using fluorescence minus one (FMO) controls. CD4+ T cell subsets were identified in terms of CD45RA and CCR7 expression. CD38 and HLA-DR were measured on gated CD4+ T cell subsets: naive CD4+ T cells (Tn, CD3+CD4+CD45RA+CCR7+), central memory CD4+ T cells (Tcm, CD3+CD4+CD45RA−CCR7+), and effector memory CD4+ T cells (Tem, CD3+CD4+CD45RA−CCR7−). The expression of CD25, CD69, and CD127 were measured on gated CD38+ Tcm, HLA-DR− Tcm, and CD4+HLA-DR+ cells. Data were collected using a BD LSRII flow cytometer (BD Biosciences) and analyzed using Flowjo software (TreeStar, USA).
3
Characterization of CD4+ T-cell Subsets
4
Multicolor Flow Cytometry of T Cells
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In brief, membrane staining of whole blood containing 0.5-1 × 10 6 white blood cells was performed with PB-conjugated anti-CD4, PE-Cy7-conjugated anti-CD25 and PerCP-Cy 5.5-conjugated anti-CD127 antibodies, all purchased from Biolegend (Biolegend, San Diego, CA, USA).
Subsequently, we used a staining set (eBioscience, San Diego, CA, USA) and AF647-labeled anti-human FoxP3 (Biolegend, San Diego, CA, USA) to perform the intracellular staining for detection of FoxP3, following the manufacturer's instructions. Flow cytometry data were acquired on a FACS Canto II instrument (BD Biosciences, USA) equipped with three lasers to allow multicolor detection with different fluorophores, using FACS DIVA software (BD Biosciences, USA).
Lymphocyte populations were selected according to the forward angle (FSC-A) and side angle (FSC-H) scattering signal, and at least 50,000 gated lymphocyte cells were detected for each sample. Dead cells were excluded by forward and side scatter characteristics, Authenticated | analuisareia@gmail.com author's copy Download Date | 9/6/15 8:37 AM and an FSC-A vs. FSC-H dot plot was used to discriminate doublets, detecting disparity between cell size vs. cell signal. Isotype control antibodies were used to help assess the level of background staining, as well as samples without staining and single stain, for each antibody.
Subsequently, we used a staining set (eBioscience, San Diego, CA, USA) and AF647-labeled anti-human FoxP3 (Biolegend, San Diego, CA, USA) to perform the intracellular staining for detection of FoxP3, following the manufacturer's instructions. Flow cytometry data were acquired on a FACS Canto II instrument (BD Biosciences, USA) equipped with three lasers to allow multicolor detection with different fluorophores, using FACS DIVA software (BD Biosciences, USA).
Lymphocyte populations were selected according to the forward angle (FSC-A) and side angle (FSC-H) scattering signal, and at least 50,000 gated lymphocyte cells were detected for each sample. Dead cells were excluded by forward and side scatter characteristics, Authenticated | analuisareia@gmail.com author's copy Download Date | 9/6/15 8:37 AM and an FSC-A vs. FSC-H dot plot was used to discriminate doublets, detecting disparity between cell size vs. cell signal. Isotype control antibodies were used to help assess the level of background staining, as well as samples without staining and single stain, for each antibody.
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