小中大展开引用
tk57ml
这个是我自己的protocol，有问题大家一起学习吧。因为我怕翻译不好，直接将我自己的protocol贡献出来吧。
2008-5-23 HSCs isolation
Mouse livers were perfused through
1. The portal vein with solution I for 5 min at 37°C at a flow rate of 7 ml/min. (35ml)
2. Each liver was then perfused solution II for 10 min at 37°C at flow rate of 7 ml/min.(70ml)
3. The liver was excised and incubated in digest solution (30ml) at 37°C for 20 min with gentle stirring.
4. Stop digestion with 10% FBS(+)EMEM 15ml
5. The incubating mixture was filtered through a mesh (pore size: 70 mm).
6. The filtrate was centrifuged at 450 g for 7 min.
7. The cell pellet was then resuspended in 5 mL of 15% OptiPrep, and loaded carefully with 5 mL of 11.5% OptiPrep, and centrifuged at 1400g for 17 min at 4°C.
8. The cell fraction in the upper layer of 11.5% OptiPrep was gently aspirated, mixed with GBSS (5ml), and centrifuged at 1400g for 10 min at 4°C.
9. the cell pellet resuspend in GBSS and was centrifuged at 450 g for 7 min
10. cell pellet was resuspended in DMEM supplemented with 10% FBS and antibiotics (105 U/l penicillin G, 100 mg/l streptomycin) 2ml
11. cultured on 24 wells plastic plates at 37°C in an incubator (5% CO2/95% air).
The purity of the cultures was evaluated by examining the characteristic stellate shape of the cells with phase-contrast microscopy and by the presence of lipid droplets by autofluorescence using an excitation light of 320 nm
Solution I
137 mM NaCl 8 g
5.4 mM KCl 0.402 g
0.6 mM NaH2PO4.2H2O 0.0936 g
0.8 mM Na2HPO4.12H2O 0.2865 g
10 mM HEPES 2.383 g
0.5 mM EGTA 0.190 g
4.2 mM NaHCO3 0.3528 g
5 mM glucose 0.901 g
pH 7.4
Total 1L
Solution II
137 mM NaCl 8 g
5.4 mM KCl 0.402 g
0.6 mM NaH2PO4.2H2O 0.0936 g
0.8 mM Na2HPO4.12H2O 0.2865 g
10 mM HEPES 2.383 g
3.8 mM CaCl2.2H2O 0.5586 g
4.2 mM NaHCO3 0.3528 g
5 mM glucose 0.901 g
180 mg/l collagenase type I
pH 7.4
Total 1L
Digest solution
solution II containing 400 mg/l pronase and 20 mg/l DNase
GBSS
NaCl, 7 g
KCl 370 mg
Na2HPO4 .2H2O 150 mg
KH2PO4 30 mg
MgCl2.6H20 376.8 mg
CaCl2.2H2O 225 mg
glucose 1 g
NaHCO3 2.27 g
Total 1 L
......
=============================================================================================================
这个方法里有个错误。吸取细胞层以后不可能用1400g这么大的离心力去沉淀细胞。这个参考文献的作者我特地写信去问过了，他承认是他们发在增补刊上的分离方法中写错了（具体文献名我不记得了，只记得文献是先发的，方法是后来增补刊的，好像是HEPATOLOGY）。应该是400g沉淀细胞，1400g梯度离心。 至于其中的灌注时间，我记得酶液好像是各4分钟，文献里有讲到。我也做这个，希望和大家多讨论
