心停止後症候群モデル動物を作成し、心拍再開後24時間で脳、肺、心臓、腸管および血漿を摘出し、速やかに凍結保存した.
保存した検体は、以下のように処理した.
1、臓器を凍結乾燥し、ジルコニアビーズを加え、チューブごと液体窒素で凍結した.
2、ボールミルを用いて20 Hz, 5 minで臓器を粉砕した.
3、2.0 mLエッペンドルフチューブに乾燥重量15 mg (0.00まで計測)以上の臓器粉末を入れた.
4、Mix Solvent (MeOH: H2O: CHCl3 =5: 2: 2)を臓器濃度が15 mg/mLとなるように添加した. (0.0 µLまで計測、蒸気圧、表面張力の誤差についてはピペッティングを行っているため、考えていない。チップの溶出性については考慮していない。)
5、20 secボルテックス後、16,000 rpm, 3 minで遠心した.
6、新しい1.5 mLエッペンドルフチューブに上清900 µLを加え、その後、MilliQ (未滅菌)を400 µL添加した.
7、16,000 rpm, 3 minで遠心し、新しい 1.5 mLエッペンドルフチューブに水層を800 µL, クロロホルム層を100 µL取得した.
8、遠心エバポレータで有機溶媒を留去した.
9、有機溶媒留去後、-80˚Cで凍結し、凍結乾燥を行った.
10、凍結乾燥サンプルに40 mg/mL Methoxyamine hydrochloride pyridine溶液 10 µL, 0.75 mg/mL d27-Myristic acid n-hexane溶液10 µLを添加した.
11、加温振盪機で30˚C, 1,200 rpm, 90 minで振盪した.
12、N-Methyl-N-trimethylsilul-trifluoroacetamide (MSTFA)+ 1% TMCSを90 µL添加し、37˚C, 1,200 rpm, 30 minで振盪した.
13、16,000 rpmで遠心し、上清を分析サンプルとした.
GS/MS解析後、主成分解析中である.
A post-cardiac arrest syndrome animal model was prepared, and the brain, lungs, heart, intestinal tract, and plasma were removed 24 hours after the resumption of heartbeat, and immediately cryopreserved. The stored samples were processed as follows.
1.The organ was freeze-dried, zirconia beads were added, and the tube was frozen with liquid nitrogen.
2.The organs were crushed at 20 Hz for 5 min using a ball mill.
3, 2.0 mL Eppendorf tubes were charged with 15 mg dry weight (measured to 0.00) or more of organ powder.
4.Mix Solvent (MeOH: H2O: CHCl3 = 5: 2: 2) was added so that the organ concentration would be 15 mg / mL. (Measure to 0.0 µL, pipette for vapor pressure and surface tension errors. (I do not think about it because I am doing it. I do not consider the elution property of the chip.)
After vortexing for 5 and 20 seconds, it was centrifuged at 16,000 rpm for 3 min.
6.Add 900 μL of supernatant to a new 1.5 mL Eppendorf tube and then add 400 μL of MilliQ (unsterilized).
After centrifugation at 7, 16,000 rpm for 3 min, 800 μL of water layer and 100 μL of chloroform layer were collected in a new 1.5 mL Eppendorf tube.
8, the organic solvent was distilled off with a centrifugal evaporator.
9.After distilling off the organic solvent, it was frozen at -80 ° C and freeze-dried.
10, 40 μL / mL Methoxyamine hydrochloride pyridine solution 10 μL, 0.75 mg / mL d27-Myristic acid n-hexane solution 10 μL were added to the freeze-dried sample.
11, shaken with a heating shaker at 30˚C, 1,200 rpm, 90 min.
12.90 μL of N-Methyl-N-trimethylsilul-trifluoroacetamide (MSTFA) + 1% TMCS was added and shaken at 37 ° C, 1,200 rpm, 30 min.
13, centrifuged at 16,000 rpm, the supernatant was used as an analytical sample.
Principal component analysis is in progress after GS / MS analysis.
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