Flow Cytometric Quantification of TCR, CD8, and pMHC

Site densities of TCR, CD8, and pMHC were measured by flow cytometry^{5 (link)} using PE-conjugated antibodies: anti-mouse Vα2 TCR monoclonal antibody (mAb) (B20.1, BD Pharmingen, San Jose, CA), anti-mouse CD4 (RM-45, eBioscience, San Diego, CA), anti-mouse CD8 (53–6.7, BD Pharmingen), anti-mouse OVA_257–264 bound H2-K^b (25-D1.16, eBioscience), anti-mouse H2-K^b (AF6–88.5, BD Pharmingen), and β2 microglobulin (S19.8, Santa Cruz Biotechnology, Dallas, TX). PE-conjugated rat IgG2a κ (eBioscience), mouse IgG2a (Santa Cruz Biotechnology), and hamster IgG3 λ₁ (BD Pharmingen) were used as isotype controls. Cells and beads were incubated with appropriate antibodies at 10 μg/ml in 100 μl of FACS buffer (PBS without calcium and magnesium, 5 mM EDTA, 1% BSA, 25mM HEPES, 0.02% sodium azide) at 4 °C for 30 min; measured the fluorescent intensity by the BD LSR II flow cytometer (BD Biosciences, San Jose, CA); and calibrated by the BD QuantiBRITE PE standard beads (BD Biosciences) to determine the site densities using the cell or bead surface area (154 μm² for a thymocyte; 140 μm² for a RBC; and 12.6 μm² for a bead)^{5 (link)}.