Fig. 5. Neuronal calcium flux responses to SDF-1. PBMC preparations or replicate granule cell cultures derived from P8 mice were loaded with 5 µM fura-2 and analyzed using a cuvette- (leukocytes) or microscope- (neurons) based calcium flux apparatus. (A) Leukocyte responses to SDF-1 are not altered by pretreatment with 20 mM KCl. (B) Granule cell neurons display calcium flux responses to 100 ng/ml of SDF-1 only after predepolarization with 20 mM KCl (tracings 1 vs. 2). (C) Although all cells produce calcium flux responses during predepolarization, pretreatment with polyclonal antibodies to CXCR4 (tracing 1) or pertussis toxin (tracing 2) abolishes neuronal responses to SDF-1. (D) Pretreatment of granule cell neurons with glutamate (100 µM) also produces maximal responses to 100 ng/ml SDF-1. Leukocyte populations of 5x10⁶ cells/ml were analysed and groups of 10-20 neurons were visualized and analyzed. Data are presented as the relative ratio of fluorescence at emission frequency of 510 nm and excitation frequencies of 340 and 380 nm and are representative of 2 separate experiments with leukocyte preparations and 4 separate experiments with neuronal preparations.