Fig. 1. Characterization of neurospheres. (A-C) Immunocytochemistry identified nestin (A), GFAP (B), S-100ß (C), ßIII tubulin (D), and the ecto-nucleotidases NTPDase2 (E) and TNAP (F) in all neurospheres investigated (representative images). Scale bars: 50 µm. (G) Analysis of ecto-phosphatase activity. Viable neurospheres were incubated in the presence 1 mM ATP, ADP, AMP, PNPP or PNP-TMP. To determine the contribution of TNAP activity, ATP hydrolysis by neurospheres was analyzed in the presence of 1 mM levamisole. No phosphodiesterase activity (PNP-TMP hydrolysis) was observed. Phosphatase activities were normalized to the activity obtained with ATP as a substrate. The 100% value corresponds to 3.2±1.1 nmoles Pi/min/100 neurospheres (mean±s.d., n=9-12, ^*P<0.05, ^**P<0.01). (H) RT-PCR products revealing the presence of mRNAs in neurosphere extracts encoding NTPDase2 and TNAP. (I) Immunoblot using total neurosphere protein (5 µg/lane) detecting protein bands of 70 and 80 kDa (arrowheads) corresponding to NTPDase2 and TNAP, respectively.