Spinal motor neurons from neonatal rats: purification, culture and identification doi: /j.issn

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1 Chinese Journal of Tissue Engineering Research October 8, 2015 Vol.19, No.42 (/ ), ( )(2013E15SF171) Hoechst 85.8% 71.6% 7.8% NF % :R318 :B : (2015) [J] (42): doi: /j.issn Spinal motor neurons from neonatal rats: purification, culture and identification Yang Lin, Liu Yang, Lv De-cheng (First Affiliated Hospital of Dalian Medical University, Dalian , Liaoning Province, China) Abstract BACKGROUND: neurons are post-mitotic cells that are difficult to survive. Isolation, purification and culture of spinal motor neurons are technical difficulties of cell culture technology. OBJECTIVE: To establish the culture system of spinal motor neurons from neonatal rats and to identify and determine the purity of spinal cord neurons. METHODS: Ventral spinal cord tissues from neonatal rats were made into cell suspension that was subjected to density gradient centrifugation and differential adherence followed by purification culture. Then, the cells on cover plates were identified and classified using immunocytochemical double staining method, and the content of cell components was calculated in combination with fluorescent Hoechst nuclear staining. RESULTS AND CONCLUSION: The cells adhered well, and the neurons accounted for 85.8%, among which, motor neurons reached 71.6%, astrocytes accounted for 7.8%, cells negative for neurofilament 200 and glial fibrillary acidic protein were 6.4%. These findings indicate that the ventral spinal cord tissues from neonatal rats combined with density gradient centrifugation and differential adherence can develop high-purity spinal motor neurons. Subject headings: Neurons; Cells, Cultured; Microglia Funding: the Natural Science Foundation of Liaoning Province, No ; the Science and Technology Project of Dalian, No. 2013E15SF171 Yang L, Liu Y, Lv DC. Spinal motor neurons from neonatal rats: purification, culture and identification. Zhongguo Zuzhi Gongcheng Yanjiu. 2015;19(42): Yang Lin, Studying for doctorate, First Affiliated Hospital of Dalian Medical University, Dalian , Liaoning Province, China Corresponding author: Liu Yang, M.D., Professor, First Affiliated Hospital of Dalian Medical University, Dalian , Liaoning Province, China Accepted: P.O. Box 10002, Shenyang

2 . 0 Introduction [1] [2-3] 20Harrison [4-5] [6] [7-9] 1 Materials and methods h SD Neurobasal B27 Biotech PLL Hoechst NF200 Sigma ChAT Chemicon TRITC IgG FITC IgG 0LYMPUS PBS 3 1mm 1 mm 1 mm 37 5%CO % 25 min 4%BSA 2 ml DMEM r/min15 min 6.8% r/min15 min 3 F1 (BSA)F2 ( )F3 ()F2 4%BSA2 500 r/min20 min 1 h (76% Neurobasal2%B27 10%FBS 10%HS 1% 1% 1% )0.4% 1 µl 9 µl L µl37 5%CO 2 3 h3 d Hoechst NF200 ChAT 4 dchat NF mol/l PBS340 g/l20 min 0.01 mol/l PBS35 min/ 3%BSA 0.5%Triton-X min 24 NF200 ( )50 µl37 1 h mol/lpbs35 min/tritc IgG(1 400) 37 1 h 0.01 mol/l PBS 35 min/ (1 500)/ ChAT37 4 h 0.01 mol/l PBS35 min/fitc IgG(1 400) 37 1 h Hoechst (1 mg/l)5 min 50% () ChAT/ ( ) NF200 ( ) NF200/ChAT ChAT NF h ISSN CN /R CODEN: ZLKHAH 6783

3 . 10 (0.12 mm 2 ) SPSS 19.0t 2 Results h ( 1) 4 d NF200() (TRITC) ChAT() (FITC) ( 2) (10 20 µm) Hoechst ( 3) Hoechst ChAT NF200 NF % ChAT71.6% ChAT ( ) 14.2% 7.8% NF % h (98.7±1.23)% (95.5± 1.51)% (92.8±1.87)% (90.6±2.25)% (P > 0.05) Discussion [10-12] [13] [14-15] [16] [17-18] 10 2 min ml 2 min 3 [19-21] 6.8% [22] r/min [23] F2 [24] P.O. Box 10002, Shenyang

4 . A B 1 24 h ( 200) Figure 1 Morphology of spinal motor neurons at primary inoculation time and 24 hours after culture ( 200) A B 24 h 2 NF200( ) ChAT( ) Hoechst( )(=50 µm) Figure 2 Spinal motor neurons were stained with neurofilament 200 (red), ChAT (green), Hoechst (blue) (scale bar=50 µm) a NF200 b ChAT c Hoechst d 3 NF200( ) ( ) Hoechst( )(=50 µm) Figure 3 Spinal motor neurons were stained with neurofilament 200 (red), glial fibrillary acidic protein (green), Hoechst (blue) (scale bar=50 µm) a NF200 b c Hoechst d 37 1 h 50 µl 3 h 85.8%71.6% GMDF B [25-27] [28] [29-30] ISSN CN /R CODEN: ZLKHAH 6785

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