독성평가를 위한 생체지표 연구 - webbook.me.go.kr

1 NIER-GP2015-157 11-1480523-002434-01 2015- i - 1I. 21. MTT Assay 22. LDH leakage Assay 43. Neutral red Assay 5II.

2 71. ROS Assay 82. NO Assay 103. GSH/GSSG Assay 12 III. 141. IL-1 / 14 1) IL-1 14 2) IL-1 152.

3 IL-6 163. IL-8 164. IL-10 175. VEGF 176. TNF- 187.

4 TGF- 19- ii - 8. 191) ELISA 202) 22IV. 26V. 29- iii - 1.

5 MTT Assay 2 2. 7 3. ROS Assay 8 4. NO Assay 10 5. GSH/GSSG Assay 12 6. 15 7. ELISA 21 8.

6 ELISA 22 9. Microplate layout 23 2015- 1 - 1. (2015)2. - - - In vivo In vitro 3. - MTT, LDH, Neutral Red Assay- ROS, NO, GSH/GSSG Assay- 8 Assay- 2 -I. in vitro.

7 MTT assay, LDH leakage assay, Neutral red assay . 3 .1. MTT Assay1) MTT assay Tetrazolium-based colorimetric (MTT) . tetrazoliumsalt[3-(4,5-dimethylthiazol-2 -yl)-2-5-diphenyltetrazolium bromide] (MTT) MTT formazan ( 500 ~ 600nm) .1),2) 1. MTT 2015- 3 -2) -PBS-MTT : MTT 2 ~ 5mg/ml PBS , tube 4 C -DMSO- 96well plate (flat bottom) - 96well plate well 1 104 ~ 1 106.

8 - well .- 37 C, 5%, CO2 incubator .- plate .- 2 ~ 5mg/ml MTT . (MTT )-37 C, 5%, CO2 incubator 4 .-MTT DMSO .- plate 10 ~ 30 .-Plate reader . ( : 500nm ~ 600nm)- 4 -2. LDH leakage Assay1) LDH (latate dehydrogenase) leakage assay LDH assay , . , ..2),4) 2) - LDH Assay kit - 96well plate (flat bottom) - 96well plate well 50 ~ 200.

9 ( : , )- Triton X-100 kit .- well .- 37 C, 5%, CO2 incubator .- 400 ~ 600g, 5 . - 96well plate LDH reaction well .- 37 C 30 . ( )-Plate reader . ( : 450nm ~ 490nm) 2015- 5 -3. Neutral red Assay1) Neutral red assay Neutral red . neutral red dye neutral red.

10 2),3),4) 2) - Neutral red Assay kit - 96well plate (flat bottom) - 96well plate well .- 37 C, 5%, CO2 incubator .- 96well plate .- Neutral red well .-2 ~ 4 37 C, 5%, CO2 incubate .- , PBS HBSS . ( )- Neutral red .- . ( - 6 - )-10 shaker .-Plate reader . ( : 540nm) 2015- 7 -II.. , DNA.