Objective: To compare the morphology, differentiation, markers, proliferation and apoptosis of murine neuroblastoma N2a cells induced with five different differential culture medium. Methods: N2a cell line was grown in complete medium[DMEM+10% fetal bovine serum (FBS)]and was replaced with five different differential medium:D1 (DMEM+1%FBS), D0 (DMEM without FBS), 30%OM (DMEM+30%Opti-MEMⅠ), D2+retinoic acid (RA) (DMEM+2%FBS+10 μmol·L-1RA) and D1+RA (DMEM+1%FBS+10 μmol·L-1RA) to allow for differentiation. The differentiation ratio, the length and numbers of neurites at different time points were measured by photographs shown in bright field, the expression of neuron markers were detected by immunostaining and Western blotting, the effects of different differential medium on cell proliferation and apoptosis were detected by EdU labeling, CCK-8 and Annexin Ⅴ/PI double staining assays. Results: The differentiation ratio of N2a cell line induced with D0 at the time of 72 h was (35.92±2.63)%, D1, 30%OM, D2+RA and D1+RA under medium density at 96 h were (10.40±2.05)%, (29.70±4.98)%, (11.37±2.56)% and (45.13±8.75)% respectively, and the expression of neuron marker βⅢ-tubulin and grouth associated protein 43 (GAP43) were up regulated in differentiated N2a cells. N2a differentiation induced with D0 had long single neurite at the time of 24 h, but the ability of proliferation was much lower than that of D10 and 30%OM, and the proportion of apoptosis and death were much higher, after 72 h of inducing, N2a cells were poor and most of them died. N2a differentiation induced with D1+RA showed long but multiple neurites, lower proliferative capacity and more apoptotic cells. N2a differentiation induced with 30%OM showed long neurites later than D0 and D1+RA, and most of the differentiated cells had two neurites. Unlike D0 and D1+RA, 30%OM showed slightly lower proliferation capacity and no significant difference of apoptosis than complete medium D10. Moreover, N2a cells could be differentiated by 30%OM for longer than 144 h under low density, the differentiation ratio and the length of the neurites reached to (56.22±6.27)% and (176.73±108.61) μm respectively. Conclusion: D0, 30%OM and D1+RA differential medium can all induce N2a differentiation to a higher ratio, D0 and D1+RA show earlier long neurite outgrowth, but lower proliferation capacity and higher apoptotic ratio than 30%OM, which is not conducive to long time culture. The differentiation induced by 30%OM can also achieve to a high differentiation ratio and long neurite under low density, moreover, it's suitable for long term culture.
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