DNA vaccines (pVAX1-OVA and pVAX1-Der-p1) were produced previously (82 (link)) and prepared by EndoFree Plasmid Maxi Kit (Qiagen). His-tagged recombinant Der p 1 protein was prepared by Ni-NTA agarose (Qiagen) and Detoxi-Gel Endotoxin Removing Gel (Thermo). Endotoxin of each vaccine was below 10 EU/mg. All reagents were from Sigma-Aldrich unless specified otherwise. Magnetic CD4+ T cell purification kit was purchased from R&D System. LTβR-Ig protein was a kind gift from Dr. Yangxin Fu (University of Chicago) and Dr. Mingzhao Zhu (Institute of Biophysics, Chinese Academy of Science). Lentiviral systems of S1p1 overexpression, CXCR3, and VLA-4 knockdown were gifts from Dr. Ying Wan (Third Military Medical University, Chongqing, China). Antibodies for CCR5 (7A4), CCR7 (4B12), CD44 (IM7), CD62L (MEL-14), CD4 (GK1.5), CD25 (PC61.5), CD69 (H1.IF3), DO11.10 TCR (KJ1-26), IL-35/IL-12p35 (4D10p35), B220 (RA3-6B2), and MHC-II (M5/114.15.2), as well as all ELISA kits, were purchased from eBioscience. Antibodies for CCR3 (TG14), CXCR3 (CXCR3-173), CCR6 (29-2L17), CCR9 (9B6), CD11a (M17/4), and CD49d (R1-2) were from Biolegend. Antibodies for S1p1 (H-60), CCR8 (c-17), and Ki67 (M-19) were from Santa Cruz. Antibody for IL35/EBI3 was from R&D System.
Cd49d r1 2
Manufactured by BioLegend
CD49d (R1-2) is a monoclonal antibody that binds to the CD49d antigen, also known as the alpha 4 subunit of the VLA-4 integrin. This antibody is used in flow cytometry and other immunological applications to identify and characterize cells expressing the CD49d antigen.
Automatically generated - may contain errors
Lab products found in correlation
Mel 14, by Thermo Fisher Scientific (1 mentions)
Detoxi gel endotoxin removing gel, by Thermo Fisher Scientific (1 mentions)
Ni nta agarose, by Qiagen (1 mentions)
Endofree plasmid maxi kit, by Qiagen (1 mentions)
Live dead fixable aqua stain, by Thermo Fisher Scientific (1 mentions)
Cd45.1 a20, by BioLegend (1 mentions)
Cd45.2 104, by BioLegend (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Lsrfortessa, by BD (1 mentions)
2 protocols using cd49d r1 2
1
Characterization of DNA Vaccines for Allergy
2
Multiparameter Flow Cytometry Analysis
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All organ-derived mononuclear cells were prepared as previously described (28 (link), 39 (link)). Fluorescein-conjugated mAbs against CD4 (GK1.5), CD8α (53-6.7), TCRβ (H57-597), CD11a (M1714), CD49d (R1-2), Tbet (4B10), IFNγ (XMG1.2), IL-10 (JES5-16E3), CD45.1 (A20), and CD45.2 (104) (Biolegend) were used. Dead cells were excluded from analysis using LIVE/DEAD Fixable Aqua Stain (Invitrogen), as per manufacturer's instructions. Both cell surface and intracellular staining was undertaken according to methods previously described (28 (link)), with all samples acquired on a BD LSRFortessa (BD Biosciences). Gating strategies used for analysis are outlined in Figures 3 , 6 . For analysis of intracellular IFNγ and IL-10, cells were stimulated for 3 h at 37°C and 5% CO2 in the presence of PMA (Sigma) and Ionomycin (Sigma) in addition to Brefeldin A (Sigma), as previously described (28 (link)).
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