小鼠代谢相关检测技术手册 - genecome.cn

1 1 1. (GTT) .. 2 2. (ITT) .. 4 3. (Pyruvate challenge test) .. 6 4. (ELISA) .. 8 5. (DEXA ) .. 11 6.. 13 7. (GSIS) .. 15 8.. 17 9.. 19 10.. 21 11. HE .. 23 12.. 25 13.. 27 14. 1(GLP-1) (ELISA) .. 28 15. (GIP) (ELISA) .. 31 16. ( glucagon ) (ELISA) .. 34 2 1. (GTT) (Cat: G6125 Sigma-Aldrich) ( NaCl) (GLUCOCARD II Series Test Meter ARKRAY) (GLUCOCARD Test Strip II ARKRAY) g 2 g ( or 2 g/kg) 2 g/kg (saline) 20% 1. ( ) 6 5 16 9 2.

2 3. 1- 2 mm 0 min 4. 30min (IP GTT) (OGTT) 5. IP GTT 1 ml g ml 6. OGTT 1 ml g ml 3 7. 1 min 8. 15 min 30 min 60 min 90 min 120 min 3 9. Excel 4 2. (ITT) (Novolin R 40 U/ml Novo Nordisk) ( NaCl) (GLUCOCARD II Series Test Meter ARKRAY) (GLUCOCARD Test Strip II ARKRAY) 30min 40% U U ( U/kg) U/kg (saline) U/ml 1.

3 ( ) 6 9 4 1 2. 1 3. 1- 2 mm 0 min 4. 30 min 5. 1 ml g ml 1 min 5 6. 15 min 30 min 45 min 60 min 3 7. Excel 6 3. (Pyruvate challenge test) (Cat: PD0462 Sangon) ( NaCl) (GLUCOCARD II Series Test Meter ARKRAY) (GLUCOCARD Test Strip II ARKRAY) 1. (gluconeogenesis) 2.

4 G 2 g ( or 2 g/kg) 2 g/kg (saline) 20% 1. ( ) 6 5 16 9 2. 3. 1- 2 mm 0 min 4. 30 min 5. 1 ml g ml 1 min 7 6. 15 min 30 min 60 min 90 min 120 min 3 7. Excel 8 4. (ELISA) Rat/Mouse Insulin ELISA kit(Cat EZRMI-13K LINCO Research) (SunriseTM TECAN) ( fast status fed status glucose- stimulation etc) 1.

5 1) 50 ul ml EP 4 C 2- 4 h 2) 4 C 4000 g 15 min 3) ( ) EP ELISA -80 C 2. (ELISA) ELISA kit Linco Rat/Mouse Insulin ELISA kit 1) kit 10 Wash buffer Milli Q H2O 1 50 ml 10 Wash buffer 450 ml Milli Q H2O 2) ELISA strips ELISA strips 4 C ELISA strips 300 ul wash buffer 3 wash buffer 9 3) QC QC 4) 10 ul Assay Buffer 10 ul Matrix Solution QC 10 ul QC 10 ul Matrix Solution 10 ng/ml 5 ng/ml 2 ng/ml 1 ng/ml ng/ml ng/ml 10 ul 10 ul Assay Buffer 5)

6 80 ul Detection Antibody 1 h ELISA strips 2 h 6) 7) 300 ul wash buffer 3 wash buffer 8) 100 ul Enzyme Solution ELISA strips 30 min 9) 10) 300 ul wash buffer 6 wash buffer 11) 100 ul Substrate Solution ELISA strips 30 min 30 min ( ng/ml) 10 12) 100 ul Stop Solution 5 min 450 nm 13) Excel 11 5.

7 (DEXA ) (Cat: 90710 Sigma-Aldrich) (50 25 g ml (Cat: A-1685 Sigma-Aldrich) 37 C 1 h 1 ) PIXImus II dual energy X-ray absorptiometry (DEXA) system (Lunar GE Medical System) 1. 2. DEXA 16 1. PIXImusII DEXA Lunar PIXI 2. DEXA 3. DEXA F6 quality control 4. QC phantom DEXA 5. F3 QC 6. QC F8 QC DEXA QC QC phantom QC ( 2-4) QC 7. ( ml/g BW) 20 g (1 ) ml 8.

8 DEXA F3 ID Enter 12 9. DEXA 10. F3 11. 3 F3 Enter lean ( / ) 12. 2 F8 DEXA 7- 10 13 6. Collagenase Type V(Cat: C9263 Sigma-Aldrich) Histopaque 1077(Cat: 10771 Sigma-Aldrich) RPMI 1640 medium( 11 mM D-glucose Cat: 22400 Invitrogen Gibco) HBSS(NaCl 114 mM KCl mM KH2PO4 mM MgSO4 mM HEPES 20 mM CaCl2 mM NaHCO3 mM adjust the pH to by NaOH) (SMZ800 Nikon) (5810R Eppendorf) 1.

9 2. RPMI 1640 medium 3. 20 ul 4. 60-80 1. 2. ( ) 4 5 ml collagenase (1 mg/ml collagenase in HBSS ice-cold) 2-3 ml collagenase 3. 50 ml collagenase 2 ml 37 C 28 min 14 4. 10 s 15 ml HBSS 5. 4 C Eppedorf 5810R 1000 rpm 30 s 6. 10 ml HBSS 1 ml 10 ml 4 C Eppedorf 5810R 1000 rpm 30 s 7.

10 5 ml Histopaque 1077 1 ml 10 ml 5 ml HBSS HBSS 5 ml Histopaque 5 ml HBSS 8. 4 C Eppedorf 5810R 2200 rpm 30 min 9. HBSS 5 ml HBSS 4 C 1000 rpm 30 s HBSS 10. 10% RPMI 1640 6 cm 20 ul 11. 10% RPMI 1640 6 cm 95% air+5% CO2 37 C 15 7. (GSIS) Krebs-Ringer buffer(NaCl 115 mM KCl mM CaCl2 mM MgSO4 mM Na2 HPO4 1 mM HEPES 10 mM) BSA fatty acid free(Cat: A8806 Sigma-Aldrich) Rat/Mouse Insulin ELISA kit Cat: EZRMI-13K LINCO Research) (glucose) (1 M) (arginine) ( M) KRB buffer (Krebs-Ringer buffer+ fatty acid free BSA) 1.