Hilyte fluor 647

The medium used throughout was RPMI 1640 medium (Sigma-Aldrich. Inc., St. Louis, MO, USA) supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin and 100 µg/ml streptomycin (hereinafter called RPMI medium). Mouse monoclonal antibodies (mAbs) specific for HTLV-I Tax (clone Lt-4), OX40 (clone B-7B5), and KLH (IgG3, clone KLH-3, Tanaka et al., unpublished) were purified in our laboratory from ascites fluids of CB.17-SCID mice carrying the appropriate hybridomas. The ascites fluid samples were subjected to ammonium sulfate precipitation followed by gel filtration with Superdex G-200 (GE Healthcare, Tokyo, Japan). mAbs were labeled with either fluorescein isothiocyanate (FITC) or HiLyte Fluor™ 647 using commercial labeling kits (Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. FITC-, PE- or PE-Cy7-labeled mouse mAbs against human CD3, CD4, CD8, CD14, CD19, CD56, and CD83 (clone H15e), and FITC-labeled goat anti-rat IgG, donkey anti-rabbit IgG, and HRP-labeled goat anti-rat IgG antibodies were purchased from BioLegend (Tokyo, Japan). Rabbit polyclonal IgG anti-rat CD83 antibody was obtained from Sino Biological (Beijing, China). Prostaglandin E2 (PGE2) was purchased from Sigma-Aldrich. Inc., (St. Louis, MO, USA).

The medium used throughout was RPMI 1640 medium (Sigma-Aldrich Inc., St. Louis, MO, USA) supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin and 100 µg/mL streptomycin (hereafter called RPMI medium). Rat and mouse monoclonal antibodies (mAbs) were purified in our laboratory from ascites fluids of CB.17-SCID mice carrying the appropriate hybridomas as described previously [17 (link)]. These antibodies were rat IgG2b mAbs anti-gp46 (clones LAT-27), rat IgG2b anti-HIV-1 p24 (clone WAP-24), mouse IgG3 anti-HTLV-I Tax (clone Lt-4). mAbs were labeled with HiLyte Fluor™ 647 using commercial labeling kits (Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. PE-labeled mouse mAbs against human CD4 were purchased from BioLegend (Tokyo, Japan). Humanized-LAT-27 (hu-LAT-27) and human-mouse chimeric antibody consisting of human IgG1 Fc and a part of mouse anti-CEA were generated in collaboration with IBL (Gunma, Japan) and the information of hu-LAT-27 will be reported elsewhere.