討論

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僻巴
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1 MTT cell viability assay 1 [45] succinate dehydrogenase MTT Kreb s cycle 2 MTT 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide DMSO 570 nm 15

2 3 MTT solution MTT Sigma Cat. M-2128 ( PBS ) 5 mg/ml 4 DMSO MERCK Germany Ord Sorenson s glycine buffer Glycine 0.1 M NaCl 0.1 M well plate 20 µl /well MTT CO 2 2~3 MTT well plate MTT MTT µl /well DMSO 25 µl /well Sorenson s glycine buffer 10 MTT MTT DMSO 5. Microplate reader BIO-RAD Model nm 16

3 µg/ml kanamycin DMEM 5 CO 2 37 ( 3.0 x well 9.0 x mm ) 10 DMEM 12 5 DMEM ELISA ( enzyme linked immunosorbent assay ) (1) Mouse monocyte chemotactic protein-1 ( MCP-1 Pharmingen Cat. # ) Coating buffer 11.8 g Na 2 HPO g NaH 2 PO 4 Q 1 ph 6.5 Assay diluent 10 FBS PBS ph 7.0 Working detector HRP ( Horseradish peroxidase ) MCP assay diluent Wash buffer 0.05 Tween-20 PBS Substrate solution Tetraamethylbenzidine 17

4 Stop solution 2 N H 2 SO 4 ( TMB ) Hydrogen peroxide ( Pharmingen Cat. 2642KK ) MCP coating buffer 100 µl 96 well ( Cat FALCON USA ) 4 12~16 wash buffer 200 µl/well assay diluent 1 wash buffer µl/well ( adhesive plate covers ) 2 wash buffer µl/well working detector 1 wash buffer µl/well substrate solution µl/well stop solution 450 nm OD (2) IL-1 TNF- ( Cat. EM-IL1B Cat. EM-TNFA Endogen USA ) 50 µl/well 50 µl/well 2 18

5 3 100 µl/well HRP µl/well TMB µl/well 450 nm OD (3) INF- ( Cat. EM-1001 Endogen USA ) 50 µl/well 2 50 µl/well µl/well HRP µl/well TMB µl/well 450 nm OD (4) IL-6 GM-CSF ( granulocyte-macrophage colony stimulating ) MIP-1 ( macrophage inflammatory protein-1 ) ( Cat. M6000 Cat. MGM00 Cat. MMA00 R D Germany ) 50 µl/well 50 µl/well 19

6 µl/well µl/well HRP µl/well TMB µl/well 450 nm OD OD MCP-1 0 pg/ml ~ 1000 pg/ml Pharmingen IL-1 0 pg~1000 pg/ml Endogen TNF- 0 pg/ml~2450 pg/ml Endogen INF- 0 pg/ml~3000 pg/ml Endogen IL-6 0 pg~1000 pg/ml R D GM-CSF 0 pg/ml~500 pg/ml R D 20

7 MIP-1 0 pg/ml~300 pg/ml R D wash buffer 1 x PBS µm sodium orthovanadate ( Na 3 VO 4 Cat. S6508 Sigma ) lysis buffer 1 x PBS + 1mM Na 3 VO Igepal CA-630 ( Cat. I-3021 Sigma ) + 1 x proteinase cocktare inhabitor ( Cat Roche Germany ) 30 wash buffer lysis buffer 500 µl ml , ( BCA protein assay kit Cat Pierce USA ) well ( Cat Greiner Germany ) 25 µl 200 µl protein assay reagent A + protein assay reagent B ( 50:1 ) nm

8 0 mg ~ 2 mg ( immunoprecipitation ) 500 µg 35 µl monoclonal anti-phosphotyrosine agarose beads ( Cat. PT-66 Sigma ) lysis buffer 1 ml , ml lysis buffer wash buffer sample buffer ( 3.6 ml Q 1 ml 0.5 M Tris-HCl 1.6 ml 10 % SDS 0.8 ml 1 M dithiothreitol 0.8 ml glycerol 0.2 ml 0.05 % bromophenol blue ) 65 µl 5 a. Final acrylamide conc. Separating gel 7.5% Stacking gel 4% dh 2 O (ml) 5M Tris-HCl ph %(w/v) SDS( µg ) Stock 40% acrylamide ( ml ) 10% APS( µl ) TEMED (µl) b. 35 µl Stacking gel ( well ) 22

9 200 V 30 c. ( transfer buffer 0.1 M Tris M glycine 5% methanol ) ( HorizBlot AE6675 ATTO Japan) Sodium Dodecyl Sulphate Poly Acrylamide Gel Electrophoresis ( SDS-PAGE ) PVDF ( polyvinylidene difluorid blotting membrane Cat.IPVH15150 Sigma ) 220 (ma) 3 PVDF 2 % BSA 1 x TBST ( 0.02 M Tris base M NaCl 0.1% Tween-20 ph 7.6 ) 4 1 x TBST PVDF x TBST 60 1 x TBST x TBST 1000 HRP 60 1 x TBST 6 5 PVDF ECL Plus 1.6 ml ( 1560 µl Solution A 40 µl Solution B Cat. RPN2132 Amersham UK ) 2 ( autoradiography cassette ) ( Kodak 23

10 BioMax Light Film Cat Kodak USA ) Total RNA 500 µm PBS 1 ml Trizol ( Cat Gibco USA ) Trizol Trizol 1.5 ml 200 µl ( rpm ) ml 500 µl / ml DEPC 260/280 nm (reverse transcription) 3 µg RNA 1.5 µl oligo(dt) 12~18 primer ( 0.5 µg/µl ) DEPC 14.5 µl 1.5 ml 24

11 µl 5 x first-strand buffer 2.5 µl dntp ( 10mM ) 1 µl DTT ( 100mM ) 1 µl superscript II Rnase H - reverse transcriptase ( 200 units/µl Gibco USA ) 1µl recombinant ribonuclease inhibitor ( RT II 40 unit /µl Gibco) 25 µl RT II 2 cdna -20 ( Polymerase Chain Reaction ) 0.2 ml 25 µl Taq DNA polymerase 10 x buffer 2.5 µl dntps mixed ( 2.5 mm,each ) 2.5 µl forward primer ( 20 µm ) 1 µl reverse primer ( 20 µm ) 1 µl cdna template 1.5 µl Taq DNA polymerase ( 5 units/µl ) 0.5 µl Q 16 µl 25 µl 0.2 ml PCR (PERKIN ELMER 2400) 25

12 µl 1.0% -20 ( MCP-1 primer forward 5` AGGTCCCTGTCATGCTTCTG Reverse 5` ACAGTCCGAGTCACACTAGTTCA ) a cdna 5x first strand buffer...8 µl oligo dt 12-18mer ( 0.5 µl/µl ).. 2 µl 10x low-dt-dntp mix... 4 µl cyanine 3 or cyanine 5 dutp 1 mm 4 µl 0.1M DTT...4 µl RNAsin...1 µl Total RNA ( label Cy5 with 40 µg or label Cy3 with 60ug ) 17 µl Total...40 µl µl superscript II enzyme µl superscript II enzyme µl EDTA ( 0.5 M ) 10 µl NaOH (1 M )

13 RNA 25 µl Tris-HCl ( 1M ph 7.5) ( microcon YM-30 columns Cat Amicon ) b purified Cy3-labeled probe 7 µl purified Cy5-labeled probe 14 µl poly da 40~60 mer 8 mg/ml 1 µl yeast Trna 4 mg/ml.1 µl COT-1 DNA 1 mg/ml.10 µl 50x Denhardt s solution. 1 µl 20x SSC..6 µl Total 40 µl µl 10 SDS 10,000 rpm 4 20 µl 30 µl 65 10~16 2 x SSC 0.05 SDS x SSC rpm 2 (GenePix4000A) 532nm 635nm 27

14 ± t (paired-t test) p 0.05 ( ) ( ratio ) log2 Cluster (filter data 80 present) hierarchical clustering Tree View 28

材料與方法

材料與方法 10-12 250-350 Sprague-Dawley rat ( SD ) 12 ( AM 8 00-PM 8 00 ) (Control group) (HS) (Heat shock group) 15 0 4 6 16 24 (PAE) ( Pulmonary air embolism group) 20 40 (25 l/min) 40 17 20 (HS&PAE) 15 4 16 20