Cd4 pe cy7 clone rpa t4

Manufactured by Thermo Fisher Scientific

Sourced in United Kingdom

The CD4-PE-Cy7 (clone RPA-T4) is a fluorescently labeled monoclonal antibody that binds to the CD4 receptor on the surface of T helper cells. It is a tool used in flow cytometry applications to identify and quantify CD4+ T cells within a sample.

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3 protocols using cd4 pe cy7 clone rpa t4

Flow Cytometry Immunophenotyping Protocol

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Flow cytometry: commercial antibodies anti-CD86-FITC (clone 2231 FUN-1), CD274 (PD-L1)-FITC (clone MIH1), CD273 (PD-L2)-PE (clone MIH-18), HLA-DR-PE-Cy7 (clone L243), IFN-γ-FITC (clone 4SB3) were purchased from BD Biosciences; CD83-PerCP-Cy5.5 (clone HB15a) was purchased from Beckman Coulter; CD80-FITC (clone MAB104), CD40-PerCP-eFluor710 (clone 5C3), CD1a-PE-Cy7 (clone HI149) and CD4-PE-Cy7 (clone RPA-T4) were purchased from eBioscience; TLR2-FITC (clone T2.5), TIM-3-PE (clone F38-2E2), IL-10-PE (clone JES3-9D7), KI-67-PE (clone Ki-67) were purchased from BioLegend; CD14-PE-DL594 (clone MEM-15), CD11c-APC (clone BU15), CD3-AF700 (clone MEM-57), CD8-PE-Dy590 (clone MEM-31) were purchased from Exbio; CD85k (ILT-3)-PE (clone 293623), CD85d (IL-T4)-FITC (clone 287219) were purchased from R&D Systems. For western blot, anti-p-p38 MAPK, anti-p-ERK1/2, anti-p-JNK/SAPK, anti-p-IκB-α, anti-IDO, anti-p-mTOR, anti-p-STAT3, anti-p-p70S6K, anti-p38 MAPK, anti-ERK1/2, anti-JNK/SAPK and anti-STAT3 Ab were purchased from Cell Signaling Technology; anti-actin was from BioLegend.

Dáňová K., Klapetková A., Kayserová J., Šedivá A., Špíšek R, & Jelínková L.P. (2015). NF-κB, p38 MAPK, ERK1/2, mTOR, STAT3 and increased glycolysis regulate stability of paricalcitol/dexamethasone-generated tolerogenic dendritic cells in the inflammatory environment. Oncotarget, 6(16), 14123-14138.

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Profiling Decidual Immune Cells

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12 samples selected for the microarray consisted of six matched maternal
and decidual mononuclear cells prepared using previously stated methods. Prior
to flow cytometric cell sorting, the following antibodies were applied: CD3 APC
(clone UCHT1, Invitrogen, UK); Live/Dead fixable red stain dye (Life
Technologies, UK), CD4 PE-Cy7 (clone RPAT4, eBioscience, UK); CD8 PerCp Cy5.5
(clone RPAT8, eBioscience, UK); CD45RA PE (clone HI100, Biolegend, UK); CCR7
FITC (clone 150503, Becton Dickinson, UK). Per maternal and decidual sample,
cells were sorted into separate CD4 and CD8 effector memory samples. RNA was
immediately isolated and extracted using the Qiagen MINI MICRO PLUS RNA easy
kit. RNA samples were immediately stored at -80 degrees. In total, 24 RNA
samples were sent to Eurofins Genomics services. Affymetrix Pico labelling kit
was used to amplify RNA, and the Affymetrix Human Gene 2.0 ST array was used to
carry out the transcriptome analysis. Microarray data accession number:
E-MTAB-5517, website URL: www.ebi.ac.uk/arrayexpress

Powell R.M., Lissauer D., Tamblyn J., Beggs A., Cox P., Moss P, & Kilby M.D. (2017). Decidual T cells exhibit a highly differentiated phenotype and demonstrate potential fetal-specificity and a strong transcriptional response to interferon. Journal of immunology (Baltimore, Md. : 1950), 199(10), 3406-3417.

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Multiparametric Flow Cytometry Analysis

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Cells were stained with Vβ4-fluorescein isothiocyanate (FITC) (TRBV29-1, clone WJF24; Beckman Coulter, Brea, CA, USA), αβTCR-phycoerythrin (PE) (clone BW242/412; Miltentyi Biotec, Bergisch Gladbach, Germany), CD3-Pacific Blue (PB) (clone UCHT1; Becton Dickinson [BD]), CD4-PeCy7 (clone RPA-T4; eBioscience, Thermo Fisher Scientific, Breda, the Netherlands), CD8-allophycocyanin (APC) (clone RPA-T8; BD), CD8-PB (clone SK1; BioLegend, San Diego, CA, USA), or RPE-conjugated NY-ESO-1_157–165 HLA∗02:01 (SLLMWITQV) pentamer (ProImmune, Oxford, UK). Samples were fixed using 1% paraformaldehyde (PFA) in PBS, measured on a FACSCanto II flow cytometer (BD, Eysins, Switzerland), and analyzed using FACSDiva (BD, Eysins, Switzerland) or FlowJo (BD, Eysins, Switzerland) software.

Kierkels G.J., van Diest E., Hernández-López P., Scheper W., de Bruin A.C., Frijlink E., Aarts-Riemens T., van Dooremalen S.F., Beringer D.X., Oostvogels R., Kramer L., Straetemans T., Uckert W., Sebestyén Z, & Kuball J. (2021). Characterization and modulation of anti-αβTCR antibodies and their respective binding sites at the βTCR chain to enrich engineered T cells. Molecular Therapy. Methods & Clinical Development, 22, 388-400.

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