Cd62l dreg 56

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CD62L (DREG-56) is a cell surface molecule expressed on various leukocyte subsets. It plays a key role in the initial tethering and rolling of leukocytes on the vascular endothelium, which is a crucial step in the recruitment of leukocytes to sites of inflammation.

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CD62L (93 citations) DREG-56 (5 citations) Anti-CD62L (clone DREG-56, (3 citations) CD62L (clone DREG-56, (3 citations) CD62L-PE (DREG-56, (2 citations) Anti-CD62L-PE-Cy5 (clone DREG-56; (2 citations) CD62L-APC (clone DREG-56), (2 citations) CD62L-BV650 (clone DREG-56), (1 citations)

4 protocols using cd62l dreg 56

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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Multicolor Flow Cytometry Analysis

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Cells were resuspended in FACS buffer and stained with antibodies specific for surface antigens. For intracellular staining, cells were incubated in fixation buffer or eBioscience fixation/permeabilization concentrate (ThermoFisher Scientific, 00-5123-43) before washing with permeabilization buffer (ThermoFisher Scientific, 00-8333-56). Anti-mouse anti-CD8α (53-6.7), CD8β (YTS156.7.7), CD44 (IM7), CD62L (MEL-14), KLRG1 (2F17KLRG1), IL-7R (SB/199), PD-1 (29F.1A12), TIGIT (1G9), Lag3 (C9B7W), T-bet (4B10), IFNγ (XMG1.2), TNFα (MP6-XT22), GzmB (GB11), Perforin (S16009A), Ki67 (16A8) and anti-rabbit (poly4064) were purchased from Biolegend. Anti-TNFα (MP6-XT22), CD98 (REA861), CD71 (REA627) and Bcl2 (REA356) were purchased from Miltenyi Biotec. Anti-TCF1 (C63D9) antibody was from CST. Anti-human CD8α (HTT8a), CD3 (OKT3), CD45RO (UCHL1) and CD62L (DREG-56) were from Biolegend. Samples were acquired using Canto II or LSR II instruments and analyzed using FlowJo software (Tree Star).

Madi A., Weisshaar N., Buettner M., Poschet G., Ma S., Wu J., Mieg A., Hering M., Ming Y., Mohr K., Ten Bosch N, & Cui G. (2022). CD8 agonism functionally activates memory T cells and enhances antitumor immunity. International journal of cancer, 151(5).

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Comprehensive T cell Phenotyping Protocol

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All samples were analyzed with an LSR Fortessa (BD Bioscience) and data were analyzed using FlowJo software (FlowJo LLC). T cell phenotype was evaluated via:Zombie Yellow Fixable Viability Kit (BioLegend), human CD45 (clone HI30, BioLegend), mouse CD45 (clone 30F11, BioLegend), CD3 (clone OKT3, BioLegend), CD4 (clone A161A1, BioLegend), CD8 (clone SK1, BioLegend), CD19 (clone HIB19, BioLegend), CD27 (clone M-T271, BioLegend), CD62L (DREG-56, BioLegend), PD-1 (clone MIH4, BD Bioscience), TIM-3 (clone F38-2E2, BioLegend), CD45RO (clone UCHL1, BioLegend), CD45RA (HI100, BioLegend).

Wang Y., Cho J.W., Kastrunes G., Buck A., Razimbaud C., Culhane A.C., Sun J., Braun D.A., Choueiri T.K., Wu C.J., Jones K., Nguyen Q.D., Zhu Z., Wei K., Zhu Q., Signoretti S., Freeman G.J., Hemberg M, & Marasco W.A. (2024). Immune-restoring CAR-T cells display antitumor activity and reverse immunosuppressive TME in a humanized ccRCC mouse model. iScience, 27(2), 108879.

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Multiparameter Analysis of PBMC and Blister NK Cells

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Multiparameter analysis of PBMC and blister NK cell phenotype was performed on an ARIA II (BD Biosciences). PBMCs and blister cells were stained with different combinations of antibodies specific to human CD3(UCHT1;Biolegend), CD8(HIT8a;Biolegend), CD16(3G8;Biolegend), CD45(HI30;Biolegend), CD56(MEM-188;Biolegend), CD62L(DREG-56;Biolegend), CD69(FN50;Biolegend), CD107a(H4A3;Biolegend), NKG2D(1D11;Biolegend), CXCR6(K041E5;Biolegend), T-bet(04–46; BD), and EOMES(WD1928;ThermoFisher). All surface staining was performed for 30 min on ice after prior incubation of cells with Human Fc block and Murine Fc block (BD) for 10 minutes on ice. Following surface staining, cells were fixed and permeabilized using Foxp3 Transcription Factor Fixation/Permeabilization Concentrate and Diluent Kit (eBiosciences, Thermo Fisher), as per the manufacturers protocol, and stained for the expression of the transcription factors T-bet and Eomes. Fluorescence minus one control stains were performed using PBMC to verify the staining specificity and as a guide for setting markers to delineate positive and negative populations. Gating was set on the live lymphocyte population using forward- and side-scatter profiles to include lymphocytic blasts, followed by single cell gating using forward- and side-scatter heights and widths, before identification of hematopoietic cells using human CD45 staining.

Nikzad R., Angelo L.S., Aviles-Padilla K., Le D.T., Singh V.K., Bimler L., Vukmanovic-Stejic M., Vendrame E., Ranganath T., Simpson L., Haigwood N.L., Blish C.A., Akbar A.N, & Paust S. (2019). Human Natural Killer Cells Mediate Adaptive Immunity to Viral Antigens. Science immunology, 4(35), eaat8116.

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