Cd45ro uchl1

Manufactured by BioLegend

CD45RO (UCHL1) is a monoclonal antibody that recognizes the CD45RO isoform of the CD45 protein. CD45 is a transmembrane protein tyrosine phosphatase that is expressed on the surface of all nucleated hematopoietic cells. The CD45RO isoform is specifically expressed on memory T cells.

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3 protocols using cd45ro uchl1

Comprehensive Immune Profiling of TILs

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TILs were stained using Live/Dead Fixable Aqua Dead Cell Stain Kit (Life Technologies) according to the manufacturer’s instructions. Cells were stained with a combination of antibodies from BD Biosciences (unless indicated otherwise), including CD3 FITC (SK7), CD4 PerCP-Cy5.5 (RPT-T4), CD8 PB (RPT-T8), 41BB (4B4–1), PD-1 (EH12.2H7, BioLegend), CTLA-4 (BNI3), ICOS (ISA3, eBioscience), OX40 (ACT35), CD45RO (UCHL1, Biolegend), CD45RA (HI100, eBioscience), CD62L (DREG-56, eBioscience), CCR7 (G043H7, BioLegend), and Ki67 (B56). For all flow cytometry assays described, data were acquired using a Canto II cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star version 7.6.5).

Haymaker C.L., Kim D., Uemura M., Vence L.M., Phillip A., McQuail N., Brown P.D., Fernandez I., Hudgens C.W., Creasy C., Hwu W.J., Sharma P., Tetzlaff M.T., Allison J.P., Hwu P., Bernatchez C, & Diab A. (2017). Metastatic Melanoma Patient Had a Complete Response with Clonal Expansion after Whole Brain Radiation and PD-1 Blockade. Cancer immunology research, 5(2), 100-105.

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Comprehensive Immune Cell Profiling

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PBMCs were stained with CD197 (G034H7) at 37°C for 20’, followed by CD4 (RPA-T4), CD8 (RPA-T8), CD69 (FN50), CD95 (DX2), CD45RA (HI100), and CD45RO (UCHL1) for 30’ at 4°C (Biolegend, San Diego, CA). Murine cells were stained with CD4 (GK1.5), CD8a (53–5.8), CD25 (PC61), CD44 (IM7), CD45 (30-F11), CD45.1 (A20), CD45.2 (104), CD62L (MEL-14), GITR (DTA-1), Sca-1 (D7), TCRβ (H57–597), TNF-ɑ (MP6-XT22) (all Biolegend) and live-dead (Life Technologies; Carlsbad, CA). Cells were fixed and permeabilized according to instructions (ThermoFisher). Human PBMCs were stained with IFN-ɣ (B27), Ki67 (Ki67), and TNF-ɑ (Mab11). Murine cells were stained with Bcl-2 (BCL-10C4) and TNF-ɑ (Mab11). Samples were acquired on an LSR-II (BD; San Diego, CA), and data were analyzed using FlowJo v10.3.1 (TreeStar; Ashland, OR).

Padgett L.E., Dinh H.Q., Wu R., Gaddis D.E., Araujo D.J., Winkels H., Nguyen A., McNamara C.A, & Hedrick C.C. (2020). Naive CD8+ T Cells Expressing CD95 Increase Human Cardiovascular Disease Severity. Arteriosclerosis, thrombosis, and vascular biology, 40(12), 2845-2859.

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Murine Immune Cell Phenotyping

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Fluorochrome-conjugated antibodies specific for mouse CD4 (GK1.5), CD8a (53–6.7), CD5 (53–7.3, isotype: rat IgG2a, κ), IFN-γ (XMG1.2), CD45 (30-F11, isotype: rat IgG2b, κ), CD326 (EpCAM, G8.8, isotype: rat IgG2α, κ), I-Ab (AF6-120.1), CD11c (N418), and FoxP3 (MF-14), and human CD3 (HIT3a), CD8a (HIT8a), CD4 (OKT4), CD45RA (Hl100), CD45RO (UCHL1), and CD62L (DREG-56) were obtained from BioLegend. Mouse CD3ε (145-2C11), CD69 (H1.2F3, isotype: Armenian hamster IgG), Ly51 (6C3, isotype: rat IgG2a, κ), and HLA-DR (L243) specific antibodies were purchased from BD. The TCR Vβ repertoire kit (IOTest Beta Mark) was purchased from Beckman Coulter. UEA I lectin was obtained from GeneTex. Thymus, spleen, and LNs from 1–2-mo-old C57BL/6, ABabDII and ABabDR4 mice were isolated. Cells were obtained by mashing the organs through a 0.45-µm cell strainer. Isolation of thymic DCs and epithelial cells was performed as published (Xing and Hogquist, 2014 (link)). In brief, thymic lobes were digested in enzyme solution (RPMI-1640 medium with 0.05% Liberase TH and 100 U/ml of DNase I) at 37°C for 20 min. Single cells were then stained with antibodies specified in the respective figure legends and analyzed by flow cytometry (FACSCanto II; BD). FoxP3 staining was performed with True-Nuclear transcription factor buffer set from BioLegend.

Chen X., Poncette L, & Blankenstein T. (2017). Human TCR-MHC coevolution after divergence from mice includes increased nontemplate-encoded CDR3 diversity. The Journal of Experimental Medicine, 214(11), 3417-3433.

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