HCM transfers hypoxic stimulation of neurogenesis to normoxic cultures. Cerebral cortical cultures were exposed to normoxia or hypoxia for 8 hours, medium was removed, and normoxia-conditioned control medium (none), whole HCM (all), or HCM fractions were added to normoxic cultures, together with BrdU and, in some cases, pFB-hrGFP, for 24–72 hours (a). Incubation for 72 hours with HCM fractions of more than 30 kDa increased the number of cells showing BrdU incorporation and GFP fluorescence (b). HCM fractions of less than 30 kDa had no effect on BrdU or pFB-hrGFP labeling, but reduced cell viability at 24–72 hours as measured by MTT absorbance (c). To characterize BrdU-labeled cells, cultures were exposed to hypoxia for 8 hours, medium was removed, and HCM was added to normoxic cultures, together with BrdU, and in some cases, pFB-hrGFP (d) for 72 hours. Some cultures were also stained with Ab’s against nestin (e) or MAP-2 (f). BrdU incorporation colocalized with pFB-hrGFP infectivity and with nestin, but not MAP-2 immunostaining. Data are mean ± SEM, n = 3 (b), mean values that varied by less than 10%, n = 3 (c), or representative fields from three experiments (d–f).