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αcd3 okt3

Manufactured by BioLegend

αCD3 (OKT3) is a monoclonal antibody that binds to the CD3 complex on the surface of T cells. The CD3 complex is essential for T cell activation and signal transduction. This product is intended for research use only and its specific applications should be evaluated by the user.

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2 protocols using αcd3 okt3

1

Assay for CD8+ T Cell Adhesion

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Cellular adhesion was performed as previously described with some modification (Bilal et al., 2015 (link); Chapman et al., 2012 (link)). Briefly, flat-bottomed 96-well plates (Thermo-Fisher) were coated with 0–10 μg of αCD3 (OKT3, Biolegend). P14 CD8 T cells were isolated by positive selection, based on Thy1.1. 5 × 106 P14 CD8 T cells were incubated on the plate for 30 min. Non-adherent cells were removed by quickly inverting the plate to empty contents. Adherent cells were stained with αCD8a-APC-Cy7 (53–6.7; Biolegend). Cells were washed twice with PBS before being imaged utilizing Licor Odyssey Infrared detector.
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2

Isolation and Culture of DCreg from Monocytes

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CD14+monocytes and CD4+CD127low T-cells were isolated from PBMCs of healthy blood donors using RosetteSep Human Monocyte Enrichment Kits and CD4+CD127low T-cell Enrichment Kits, respectively (Stem Cell Technologies). CD14+monocytes were further purified to >97% purity by magnetic separation with anti-CD14 conjugated microbeads (Miltenyi Biotec). Memory CD4+ Th (T helper) and Tregs were obtained by sorting pre-enriched T-cells with fluorescently labeled mAbs against CD4, CD25, CD127, CD45RA, CD45RO and lineage markers along with propidium iodide (PI; Life Technologies). Th were defined as PI-Lin-CD4+CD45RA-CD45RO+CD127+CD25-/low, and Tregs were defined as PI-Lin-CD4+CD127low CD25+. Cells were sorted with a BD FACSAria IITM after staining, as above. Monocytes (1×106) were cultured with allogeneic Th and Tregs at a 10:1:1 in 12-well plates with 50ng/ml αCD3 (OKT3, BioLegend). HLA-DR+CD2- DCreg were sorted out by flow cytometry on day 4 of culture. DCreg were stimulated with 1μg/ml LPS. After 18 hr, cells were washed 3x in PBS and HLA-DR+CD2- DC were purified by FACS and incubated with allogeneic naïve CD4+ T-cells (105/well) at a 1:2 DCs to T-cell ratio in the presence or absence of 2μg/ml anti-TGFβ. After 6 days, responder CD4+ T-cells were analyzed for CD25 and Foxp3 expression.
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