µ µ µ κ 17
18
19
20
µ µ µ 21
H 2 DCF-DA H 2 O 2 H 2 DCF-DA 酶 esterase H 2 DCF-DA deacetylation H 2 DCF H 2 DCF H 2 DCF DCF H 2 DCF DCF Bae et al., 1997; Vanden et al., 1997; Atlante et al., 1997; Wang et al., 2003 relative fluorescent unit, rfu 2,7-dichloridihydrofluoresceinH 2 DCF-DA : Sigma/molecular probe 1. 24 500l 2. 20 mm H 2 DCF-DA 24 cytofluormetry excitation 485 nm emission 530 22
酶 酶 23
酶 酶 酶 酶 24
酶 酶 酶 25
µ µ 26
κα κα κα µ µ µ µ 27
2. Coomassie Brilliant Blue G-250 DDI 14 µ µ µ 28
µ µ 29
µ µ µ 30
31
Wang et al., 2000 R&D ELISA kitduo Set multiple antibody sandwich principle, ELISA 96 IL-6, MIP IL-6, MIP 酶 IL-6, MIP peroxidase-conjugated polyconal anti-il-6, anti-mip rat IL-6, MIP 酶 450 nm IL-6, MIP IL-6, MIP 32
1. phosphate bufferpbs 1X- ph 7.2-7.4 2. wash buffer- 0.05 % Tween 20 PBS 3. Block buffer- 1 % BSA5 % sucrose PBS 4. Reagent diluent- 1 % BSA PBS 5. Substrate solution- TetramethylbenzidineTMB 6. Stop solution- 2 N H 2 SO 4 7. ELISA kit kit Reagent diluent 1. kit PBS 100l 96 96 overnight 2. wash buffer 300l 3 3. block buffer100l 1 4. 2 5. 100l 96 2 6. 2 7. 100l HRP Reagent diluent 1 33
8. 2 9. 100l Streptavidin-HRP1:200 10. 2 11. 100l substrate solution TMB 20 12. 50l stop solution2 N 2 HSO 4 30 ELISA reader 450 nm ELISA reader 34