THE EFFECT OF MACROPHAGE CONDITIONED MEDIUM ON THE SURVIVAL OF CEREBELLAR CORTICAL NEURON
GUO WAN-HUA(Department of Histology & Embryology, Sun Yat-Sen University of Medical Sciences, Guangzhou 510089)ZHOU MING-HUA (CHAU, R.M.W.)(Department of Anatomy, Faculty of Medicine, University of Hong Kong, Hong Kong)
Macrophage culture was prepared and its conditioned medium (MφCM) was collected to measure its neurotrophic activities by a rapid automated colorimetric microassay. Cerebel-lar cortical neuron was dissociated from 7-day-old Sprague-Dawley rats and seeded in poly-L-ornithine coated 96-well culture plates at a density of 1×104, 2×104, 4×104, 6 ×104, 8×104, and 1×105 cells per well. The culture medium was MEM containing 6% fetal calf serum. Various capacities and molecular weight fractions of MφCM were added to the medium in experimental groups. The period of culture lasted 24, 48 or 72 hours. A yellow tetrazolium derivative, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bronide) was added to each culture at the last 4 h of culture. MTT was taken up selectively by active neuron and converted to a blue formazan product. The amount of blue color development could be rapidly quantified using an automatic microplate reader. The resulting optical density was directly proportional to the number of active neurons. Our experimental results show that the number of active neurons at 48 and 72 h of culture markedly decreases as compared with that at 24 h of culture, whereas MφCM plays a role in supporting the neuronal survival and enhancing the neuronal activity, especially at a density of 1× 105 cells and a capacity of 10μl MφCM per well (p0.05). The effects of various molecular weight fractions of MφCM on neuronal growth and activity are different in which MφCM fraction of 10KD is significantly better than that of 10KD.