Anti cd3ε

Calcium flux measurements were performed as previously described. Briefly, thymocyte cell suspensions from SHP1^fl/fl or CD4-Cre SHP1^fl/fl mice were made and rested for 60 min at 37°C. One of the samples was stained with Cell Trace Violet (CTV) for 10 min at room temperature while the other sample received equal amounts of vehicle-only control (DMSO). Cells were then washed and equal number of cells were mixed together at a density of 2×10⁶ cells/ml in R10 media (RPMI1640, 10% (v/v) FCS, 2mM L-glutamine, 0.05mM 2-mercaptoethanol and 0.05mg/ml gentamicin sulfate). The mixed thymocytes were incubated with the calcium indicator Indo-1 AM (Molecular Probes; 1mM final concentration) for 15 min at room temperature. Cells were washed once in R10 and stained for 15 min at room temperature with fluorescently conjugated antibodies and biotinylated anti-CD3ε (Tonbo Biosciences; 145-2C11) antibody. Cells were washed once in cHBSS (Ca²⁺ and Mg²⁺ free Hank’s balance salt solution supplemented with 1% (v/v) FCS, 1mM MgCl₂, 1mM EGTA and 10mM HEPES, pH 7.3) and resuspended in cHBSS. Samples were warmed to 37°C for 2 min and then analyzed on a LSRII (Becton Dickinson). CaCl₂ (2mM) was added 30 seconds into analysis followed by streptavidin (10 mg/ml) at 60 seconds to activate the samples.