Soluble biotinylated CD1b monomers were produced in lentivirus-transduced HEK293 T cells by the National Institutes of Health Tetramer Core Facility (Emory University, Atlanta, GA) and tetramerized with fluorescently labeled streptavidin. In brief, human β-2-microglobulin and the extracellular domain of CD1b were cloned into the expression vector pCMJJ4 (gift from J. Jacob, Emory University, Atlanta, GA). Lentiviral particles were made in a second generation packaging system (Naldini et al., 1996 (link)). The light and heavy chains are expressed under control of the CMV promoter and are separated by the 2A-TaV peptide to generate two separate proteins from a single mRNA. The chains are followed by a C-terminal acidic or basic leucine zipper which stabilizes the complex and is used for affinity purification using the 2H11 monoclonal antibody (E. Reinherz, Harvard, Boston, MA). Purified monomers were enzymatically biotinylated at the BirA site at the C terminus of the heavy chain. Monomer purity and composition were confirmed by PAGE, and biotinylation was confirmed by streptavidin bead pulldown assay. Functional activity was assayed by affixing biotinylated monomers at final concentration of 5 µg/ml onto 96-well streptavidin plates (Thermo Fisher Scientific) in PBS, pH 7.4, for 24 h at 37°C. Lipid antigens were sonicated in PBS for 2 min, added to the wells, and incubated for 24 h at 37°C before washing three times with 200 µl/well sterile PBS. 105 LDN5 cells were added in a total volume of 200 µl T cell medium per well (RPMI). The plate was incubated for 24 h at 37°C after which culture supernatants were collected for HT2 bioassay.