*4.7. Ca2*<sup>+</sup> *Signaling Measurements*

The intracellular Ca2<sup>+</sup> levels were measured using a flow cytometer with the Fluo-3 indicator as described before [40,41]. Briefly, single-cell suspensions were prepared from the lymph nodes of the arthritic mice and suspended in RPMI supplemented with 5% FBS and 2M CaCl2 (1 <sup>×</sup> 10<sup>6</sup> cells/mL). Cells were loaded with Fluo-3-AM (Invitrogen, Carlsbad, CA, USA) for 30 min at 37 ◦C in humidified air with 5% CO2. Using a flow cytometer, we gated on the lymphocytes based on FSC/SSC parameter distribution. The Fluo-3 fluorescence which is proportional to the intracellular Ca2<sup>+</sup> level [40] was detected in the FL1 channel. The baseline Fluo-3 fluorescence was measured for 1 min, then cells were stimulated with anti-mouse-IgM, IgG, or anti-mouse CD3 followed by secondary anti-hamster antibodies, measurements for five and a half minutes. Data were analyzed by the Cell Quest software (BD Biosciences, San Jose, CA, USA). The FL1 mean fluorescence intensity values were calculated along the time axis of the plots, and these were divided by the baseline fluorescence value, thereby the Ca2<sup>+</sup> signal was expressed as FL1 change [41]. Please note that all Ca2<sup>+</sup> measurements were performed using unsorted lymph node cells and the B or T cell activation was only distinguished based on the specific nature of the activation: anti-mouse-IgM or IgG activates only B cells through the BcR, whereas anti-CD3 cross-linking activates T cells selectively.
