Flow cytometry was performed with a FACS Canto II (BD biosciences) as previously described [22] (link), [23] (link), [25] (link), [26] (link), and the obtained data were analyzed with Cell Quest software (BD biosciences) and FlowJo software (Tree Star, Inc.). For flow cytometry analysis, anti-CD45-PE (HI30; Biolegend), anti-CD3-APC-Cy7 (HIT3a; Biolegend), and anti-CD4-APC (RPA-T4; Biolegend) antibodies were used. Hematometry was performed with a Celltac α MEK-6450 (Nihon kohden, Co.) as previously described [23] (link), [25] (link), [26] (link).
Anti cd4 apc rpa t4
Manufactured by BioLegend
Anti-CD4-APC (RPA-T4) is a fluorescently labeled antibody that binds to the CD4 protein, which is expressed on the surface of helper T cells. It can be used for the detection and enumeration of CD4+ T cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd45 pe hi30, by BioLegend (2 mentions)
Anti cd3 apc cy7 hit3a, by BioLegend (2 mentions)
Celltac α mek 6450, by Nihon Kohden (2 mentions)
Cellquest software, by BD (2 mentions)
Facscanto 2, by BD (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Anti cd14 pe, by BD (1 mentions)
Lsr 2, by BD (1 mentions)
Anti cxcr4 apc, by BioLegend (1 mentions)
Anti ki67 pe b56, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
Facsjazz, by BD (1 mentions)
Brilliant stain buffer, by BD (1 mentions)
Anti cd3 v500 ucht1, by BD (1 mentions)
Human trustain fcx blocking solution, by BioLegend (1 mentions)
Phosflow perm buffer 3, by BD (1 mentions)
4 protocols using anti cd4 apc rpa t4
1
Flow Cytometry and Hematometry Analysis
2
Flow Cytometry Analysis of HIV-Infected Cells
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Macrophages were harvested for flow cytometry from Savillex PTFE jars by gentle pipetting. TZM-gfp cultures (including co-cultures) were washed with cold PBS, then trypsin-harvested with gentle pipetting. Cells were pelleted by centrifugation at 200xg, washed twice in FACS buffer (0.1% BSA in PBS), then resuspended in Cytofix/Cytoperm (BD) for 1 h at room temperature. Cells were again washed with FASC buffer, filtered through a 70 µM nylon strainer (Fisher), and stained with conjugated antibodies in 100uL final volume in the dark. Stained samples were washed and run on a BD LSRII instrument. Data were analyzed using FlowJo software. Antibodies for flow cytometry included: Anti-p24-RD1 (KC57, Beckman Coulter), Anti-CD14-PE (M5E2, BD), Anti-CCR5-PE (2D7, BD), Anti-CD4-APC (RPA-T4, BioLegend), Anti-CXCR4-APC (12G5, BioLegend).
3
Flow Cytometry and Hematometry Protocol for Cell Analysis
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Flow cytometry was performed with FACS Calibur (BD Biosciences) and FACSJazz (BD Biosciences) as previously described [11 (link),12 (link),22 (link)–24 (link)], and the obtained data were analysed with Cell Quest software (BD Biosciences) and FlowJo software (Tree Star, Inc.). For flow cytometry analysis, anti-CD45-PE (HI30; Biolegend), anti-CD3-APC-Cy7 (HIT3a; Biolegend), anti-CD4-APC (RPA-T4; Biolegend), anti-CD25-APC (BC96; eBioscience), and anti-Ki67-PE (B56; BD Biosciences) antibodies were used. Hematometry was performed with a Celltac α MEK-6450 (Nihon Kohden Co.) as previously described [11 (link),12 (link),23 (link),24 (link),49 (link)]. Live cell sorting was performed using FACSJazz (BD Biosciences) according to the manufacture's procedure. The purity of each population was >94% (see also S7 Fig ).
4
Intracellular Phosphorylated Protein Detection
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For intracellular phosphorylated protein detection, cells were re-stimulated with human anti-CD3/CD28/CD2 (StemCell, Vancouver, Canada, 1:100) for 6 or 24 h at 37 °C in 5% CO2 incubator prior to staining. Nuclear permeabilization was performed with BD Phosflow™ Perm Buffer III (BD Biosciences) on ice for 30 min. Cells were then washed twice with PBS. Extracellular and intranuclear markers were stained at room temperature for 30 min with the following monoclonal antibodies raised against human proteins prepared in permeabilization buffer, Human TruStain FcX blocking solution (Biolegend), and Brilliant Stain buffer (BD Biosciences): anti-CD4 APC (RPA-T4, Biolegend), anti-CD8𝛼 BV605 (SK1, Biolegend), anti-CD3 V500 (UCHT1, BD Biosciences), anti-CD45RO APC/Fire 750 (UCHT1, Biolegend), anti-GATA3 PE (L50-823, BD Biosciences), anti-OX40 BV711 (Ber-ACT35, Biolegend), anti-ICOS AF700 (C398.4A, Biolegend), and anti-HLA-DR BV395 (G46-6, BD Biosciences). Cells were washed and resuspended in PBS for acquisition. For some experiments cells were acquired with Amnis® ImageStream®XMark II system and analyzed with IDEAS® Version 6.2 (Image Data Exploration and Analysis Software) following the nuclear colocalization method pre-installed in this software.
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