For cytokine detection, PBMCs and CD45RA−CD4+ T cells were activated with 50 ng/ml Phorbol 12-myristate 13-acetate (PMA) (Sigma), 500 ng/ml ionomycin (Sigma), and GolgiPlug (1 μg per 1×106 cells, BD Biosciences) for five hours. Cells were washed in staining buffer (1% FBS and 0.1% sodium azide in PBS) and stained with following antibodies: anti-human CD4 PacificBlue (RPA-T4, BD Biosciences), CD4 PerCP-Cy5.5 (OKT-4, eBioscience), CD8 PacificBlue (RPA-T8, BD Biosciences), GM-CSF PE (BVD2-21C11, BD Biosciences), IL-17A Alexa Fluor 488 (eBio64DEC17, eBioscience), IFN-γ APC (4S.B3, eBioscience), T-bet PerCP-Cy5.5 (eBio4B10, eBioscience), RORγt PE (B2D, eBioscience), IL-4 FITC (8D4-8, eBioscience), GATA3 PE (TWAJ, eBioscience), Foxp3 FITC (206D, Biolegend), IL-22 APC (IL22JOP, eBioscience), CCR10 PE (1B5, BD Biosciences), CD45RA PE (HI100, BD Biosciences), and CD45RO PE-Cy7 (UCHL1, BD Biosciences). Cells were fixed and permeabilized with Caltag Fix/Perm reagents (Invitrogen) following the manufacturer’s instructions. Data were acquired on a FACSAria (BD Biosciences) and analyzed using FlowJo software (TreeStar).
Cd8 pacificblue rpa t8
Manufactured by BD
The CD8 PacificBlue (RPA-T8) is a fluorescently-labeled antibody for the detection of CD8 positive cells in flow cytometry applications. It binds to the CD8 cell surface marker, which is expressed on a subset of T cells and other immune cells. The antibody is conjugated with the PacificBlue fluorochrome, allowing for its detection using appropriate flow cytometry instrumentation and analysis software.
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Lab products found in correlation
Facsaria, by BD (1 mentions)
Golgiplug, by BD (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Gm csf pe bvd2 21c11, by BD (1 mentions)
Gata3 pe twaj, by Thermo Fisher Scientific (1 mentions)
Foxp3 fitc, by BioLegend (1 mentions)
Cd45ro pe cy7 uchl 1, by BD (1 mentions)
Caltag fix perm reagents, by Thermo Fisher Scientific (1 mentions)
Cd27 bv605, by BioLegend (1 mentions)
Cd3 h7apc sk7, by BD (1 mentions)
Aqua dye, by Thermo Fisher Scientific (1 mentions)
Cd20 bv570, by BioLegend (1 mentions)
Pd1 bv711 eh12.2h7, by BioLegend (1 mentions)
Tnf α pe cy7, by BD (1 mentions)
Lsrfortessa cell analyzer, by BD (1 mentions)
Streptavidin pacific blue, by Thermo Fisher Scientific (1 mentions)
Tnf α pe, by BD (1 mentions)
Summit data acquisition and analysis software, by Agilent Technologies (1 mentions)
Cd3 pacific blue sp34 2, by BD (1 mentions)
Cd49d 9f10, by BD (1 mentions)
3 protocols using cd8 pacificblue rpa t8
1
Cytokine Detection in T Cell Subsets
2
Multiparametric Flow Cytometry of Tonsil Cells
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The following titrated, conjugated antibodies were used for flow cytometry analysis: CD3-H7APC (SK7, BD Biosciences), CD4-BV650 (SK3, BD Biosciences), CD8-Pacific Blue (RPA-T8, BD Pharmingen), CD19-FITC (J3-119, Beckman-Coulter), CD27-BV605 (O323, Biolegend), CD45RO-Cy5PE (UCHL1, BD Pharmingen), PD-1-BV711 (EH12.2H7, Biolegend), CD57-AF594 (NK-1, Novus Bio), IgD-PE (Goat polyclonal, Southern Biotech), CD20-BV570 (2H7, Biolegend), CD38-BV786 (HIT2, Biolegend). 1-2×106 tonsil-derived cells were thawed and rested for 2h in a cell culture incubator before staining. Cells were washed with PBS, BSA (0.5%), incubated (5 min, RT) with a viability dye (Aqua-dye, Invitrogen) and surface stained with titrated amounts of antibodies for 30 min at room temperature. After washing, cells were resuspended and fixed with 1% paraformaldehyde.
3
Cytokine Analysis by Flow Cytometry
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The following Abs were used for culture or surface and intracellular cytokine staining for flow cytometry: CD28 (CD28.2, BD), CD49d (9F10, BD), CD3-Pacific blue (SP34-2,BD), CD4-BV510 (L200, BD), CD8- Pacific blue (RPA-T8, BD), IFN-γ-APC (4S.B3, BD), IFN-γ Brilliant Violet 711 (4S.B3, Biolegend), TNF-α-PE (Mab11, BD), TNF-α-PE-Cy7 (Mab11, BD), IL-17-PE (eBio64CAP17, eBioscience), IL-22-biotinylated (anti-human IL-22, RD), Streptavidin-Pacific blue (invitrogen), Perforin-biotinylated (Pf-344, Mabtech), Caspase 3-AF647 (C92-605,BD), anti-Vγ2-FITC (7A5, Pierce).
After staining, cells were fixed and subjected to analysis on flow cytometer of BD LSRFortessaTM Cell Analyzer. Lymphocytes were gated based on forward- and side-scatters, and at least 40,000 gated events were analyzed using Summit Data Acquisition and Analysis Software (Dako Cytomation).
After staining, cells were fixed and subjected to analysis on flow cytometer of BD LSRFortessaTM Cell Analyzer. Lymphocytes were gated based on forward- and side-scatters, and at least 40,000 gated events were analyzed using Summit Data Acquisition and Analysis Software (Dako Cytomation).
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