Furthermore, the synthetic hapten molecule Cy3 contains two modified indole organizations joined by polymethine bonds. al., 1988; Schild et al., 1994; Number 1B). We then identified Cy3-specific TCRs on a single cell level by sorting these cells and sequencing their TCR genes. 58– cells expressing Cy3-specific TCRs bound Cy3-ovalbumin (Cy3-OVA), Cy3-bovine serum albumin (Cy3-BSA), Cy3-MCC-streptavidin (moth cytochrome C (MCC)-derived peptide, Cy3-labeled in the N-terminus, biotinylated in the C-terminus, and tetramerized with streptavidin), but not FITC or APC labeled OVA, nor PE-MCC peptide/streptavidin (Number 1C, Number 1figure product 1; Table 1). Moreover, Cy3-MCC-streptavidin staining of a Cy3-specific TCR NX6/58– was inhibited from the inclusion of Fab fragments of an anti-Cy3 antibody (clone A-6; Santa Cruz Biotechnology) (Number 1D). In addition, NX6/58– cells were triggered by plate-bound Cy3-OVA, but not unmodified OVA (Number 1E). Binding of the soluble form of a Cy3-specific TCR (NX6) to Cy34SAv can be shown by surface plasmon resonance (Biacore) with an apparent KD of 78.2 nM (Number 1F). We also examined the affinity of Cy34SAv binding to NX6 indicated on 58– cells. Scatchard analysis showed an apparent nanomolar KD (1.8 nM) having a half-life of 26 min (Number 1G). Taken collectively, these results show that Cy3 is an antigen of T cells, identified directly by specific TCRs. Table 1. TCR sequences of Cy3 and NP-specific TCRs DOI: http://dx.doi.org/10.7554/eLife.03609.006 expression). Upper panel shows genes expressing higher (p < 0.001) in Cy3+ cells than that in Cy3? cells. Middle panel shows non-varying genes. Bottom panel shows genes expressing lower (p < 0.001) in Cy3+ cells than that in Cy3? cells. (C) Thy1.1 expression about T cells from mouse splenocytes and PE staining of T cells from B6 splenocytes; (C) staining of 58-- cells expressing an NP-specific TCR, 1G9, with NP43-CGG-Cy5 or CGG-Cy5, showing staining in relation to TCR manifestation (remaining) or like a histogram (right); (D) staining of 58-- cells expressing an NP-specific TCR, 1E3, with NP43-CGG-Cy5, NP26-BSA-Cy5, or BSA-Cy5 (left) and NP67-PE alone, NP67-PE with a 20-fold molar excess of anti-NP Fab, or PE (right). (E) IL-2 production by 1E3/58-- cells activated by the indicated amount of plate-bound NP25-KLH, KLH (light gray bars), or 0.1 g/ml anti-CD3. (F) Sensorgram and constant state analysis of NP43-CGG (0C7 M) binding to soluble 1G9 TCR measured by surface plasmon resonance. Apparent KD was determined by steady state analysis of SPR measurements (circles). Equal concentrations of un-modified CGG were tested (squares), as well as NP43-CGG with a PE-specific TCR, MA2 (triangles). DOI: http://dx.doi.org/10.7554/eLife.03609.008 NP conjugated to a fluorescent protein, phycoerythrin (PE), is routinely used to identify NP-specific B cells in FACS analysis. We found that NP-PE stained 0.14% of splenic T cells of normal mice (left panel), but not G8/ TCR transgenic cells (middle panel). Consistent with the observation that PE is usually a T cell antigen (Zeng BRAF inhibitor et al., 2012), we found 0.03% of splenic T cells stained with PE under the same staining conditions (right panel). After accounting for background staining and for PE staining, we estimated that 0.1% of BRAF inhibitor total T cells could be NP-specific (Determine 3B). We further recognized NP-specific TCRs on a single cell level. Expressing NP-specific TCRs in 58– cells enables these cells to be stained with NP-CGG-Cy5, but not CGG-Cy5 BRAF inhibitor (Physique 3C; Table 1). Further investigation showed 58– cells expressing NP-specific TCRs could also be stained with NP-BSA-Cy5 and NP-PE, but not with BSA-Cy5 or PE (Physique 3D, left panel). In addition, NP-PE staining was inhibited by the Goat Polyclonal to Mouse IgG inclusion of Fab fragments of an anti-NP antibody (clone H33L; G. Kelsoe) (Physique 3D, right panel). Furthermore, 58– cells expressing NP-specific TCRs produced IL-2 in response to plate-bound NP-keyhole limpet hemocyanin (NP-KLH), but not plate-bound KLH in a dose-dependent manner (Physique 3E). The observations that only molecules made up of the NP conjugation stain NP-specific TCR-expressing cells, that NP-conjugate staining is usually blocked by an anti-NP Fab, and that an immobilized BRAF inhibitor NP-conjugate can activate NP-specific T cells indicate that NP is usually recognized directly by specific TCRs. Indeed, direct binding between soluble NP-specific TCRs (1G9) and NP-conjugates was also exhibited using surface plasmon resonance (Physique 3F). The measured apparent KD for the conversation between NP43-CGG and the 1G9 TCR was 0.66 M. NP43-CGG exhibited no binding to the PE-specific TCR, MA2 (Zeng et al., 2012), and CGG did not bind 1G9 (Physique 3F). Taken BRAF inhibitor together, these results.