Within the first ten minutes after contact to the astrocytes, we defined high- and low-responder T cells showing an increase in [Ca2+] 300 nM (high-responder) or < 300 nM (low-responder). step MMP2 from slowly scanning to immotile rounded T cells. Thus, we recorded in parallel changes in [Ca2+]cellular shape, and motility of the T cells following contact to astrocytes. The role of NAADP was assessed by blocking NAADP action using the recently validated small-molecule NAADP antagonist BZ194 (Dammermann et al., 2009; Cordiglieri et al., 2010). Experimental Details Materials Fura-2/AM was purchased from Calbiochem. DMSO and probenecid were supplied by Sigma. Fibronectin WHI-P 154 was obtained from Invitrogen. BZ194 was synthesized as described (Dammermann et al., 2009). Antigens Antigen specific T cell clones were specific for guinea pig myelin basic protein (MBP). MBP was purified from guinea pig brains as reported (Eylar et al., 1979). Generation and Culturing of T Cells Rat antigen-specific T cell clones were obtained from lymph node preparations of Lewis rats immunized with MBP. Stimulation, expansion and culture of specific rat T cells were conducted under conditions as described (Flgel et al., 1999). Analysis of [Ca2+]of the T cells. For every tenth fluorescence image a transmitted light image was obtained to follow the interaction of T cells with astrocytes. Time-point zero is defined as first visible contact between a T cell and an astrocyte, as assessed by bright field microscopy. In the absence of the specific antigen, the TMBP cells remained predominantly quiescent without much alteration of [Ca2+](Fig. 1(A), images and upper panel). Within the first ten minutes after contact to the astrocytes, we defined high- and low-responder T cells showing an increase in [Ca2+] 300 nM (high-responder) or < 300 nM (low-responder). Over 90% of the T cells stayed below a [Ca2+]of 300 nM in the absence of MBP (Fig. 1(A), images and upper panel). Although T cells added to astrocytes were more or less spherical, their shape changed constantly WHI-P 154 during the experiment. Several pseudopodia reached out indicating a continuous search for specific antigen. This process was quantitatively analysed using the shape index, where an ideal circular shape corresponds to a shape index of 1 1 and any deviation increases this value (Fig. 1(A) middle panel (Donnadieu et al., 1994); for details see Methods Section). In addition, T cells were still mobile and tended to move around rather than staying in contact with one astrocyte (Fig. 1(A) lower panel). In contrast, when TMBP cells recognized MBP-pulsed astrocytes, within tens of seconds the majority of the TMBP cells displayed high [Ca2+]responses (Fig. 1(B), images and upper panel). These high-responder TMBP cells started to round up immediately after onset of the Ca2+-signal (Fig. 1(B), middle panel) and showed strong immobilization (Fig. 1(B), lower panel). Immobilization led to a stable interaction with astrocytes for the duration of the measurement indicating full formation of IS. Open in a separate window Figure 1 Ca2+ signalling, shape index and motility of TMBP cells during immune synapse formation. Astrocytes were pulsed (A) or not (B) with MBP. Resting rat TMBP cells were added to the astrocytes and Ca2+-signalling, shape WHI-P 154 index and motility were analysed. Top: Transmitted light and colour-coded images showing single cell analyses of representative T cells. T cells are highlighted by green lines in the transmitted light images. Length scale = 10 300 nM in the first ten minutes. This majority of cells, the typical low-responder TMBP cells, were low in [Ca2+](Fig. 2(A), upper panel), displayed an increased shape index over time (Fig. 2(A), middle panel), and showed motilities between 2 and 3 300 nM in the first ten minutes after contact (Fig. 3, pie diagrams and A, upper panel). Although BZ194 shifted the percentage of high-responders from 70% down to 44% (Fig. 3, pie diagrams), it.