【求助】求助

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标题:【求助】求助

ukonptp[使用道具]
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发表于 2015-10-20 11:18 资料个人空间短消息加为好友

小中大

下周开始做小鼠的HSCs 原代培养，有人做过或正在做吗？

如题
想找人学习。

ukonptp[使用道具]
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发表于 2015-10-20 11:18 资料个人空间短消息加为好友

小中大

Livers of 1-year-old male Wistar rats (450-500 g body mass, Interfauna), fed ad libitum on a stock diet, were perfused first with a Ca2+/Mg2+-free solution (137 mM NaCl, 5.4 mM KCl, 0.6 mM NaH2P04 . 2H20, 0.8 mM NaHPO4.12H20, 10 mM Hepes, 0.5 mM EGTA, 4.2 mM NaHCO3, and 5 mM glucose, pH 7.4) for 10 min at 37°C and next with 0.05% collagenase solution (137 mM NaC1, 5.4 mM KC1, 0.6mM NaH2PO4.2H20, 0.8mM Na2HPO4.12H20, 3.8mM CaCl2, 10mM Hepes, 4.2mM NaHCO3 and 500 mg/l collagenase, pH 7.4) for 30 min at 37°C. The flow rate was 10ml/min. The digested liver was excised, dispersed in Ca2+/Mg2+-free solution and filtered through gauzes. Residual hepatocytes were removed twice by a low-speed centrifugation (50 g, 4"C, 2 min). The non-parenchy-ma1 cells were pelleted by centrifugation (450 g, 4"C, 10 min). A stellate-cell-enriched fraction was obtained by the use of centrifugation with a triple-layered (9, 11, 17%) Nycodenz cushion (1400 g, 4"C, 20 min). The cells in the upper layer were washed by centrifugation (450g, 4"C, 10min) and suspended in DMEM (GIBCO) supplemented with 10% fetal-calf serum (Boehringer Mannheim) and antibiotics (105U/1 penicillin G, 100 mg/l streptomycin, and 2.5 mg/l amphotericin Black Eye. After plating, the culture medium was changed every other day.
这个是别人论文中的方法。请问小鼠的操作哪些与大鼠不同？谢谢

ukonptp[使用道具]
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发表于 2015-10-20 11:20 资料个人空间短消息加为好友

小中大

没有人吗？那我换个问题，
大鼠的HSC和小鼠的HSC大小差很多吗？我isolation的时候如何选择filter的pore大小？大鼠文献上用100微米的，小鼠谁知道用多少的？谢谢

ukonptp[使用道具]
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发表于 2015-10-20 11:21 资料个人空间短消息加为好友

小中大

还是没有人吗？
再换一个问题
1. livers were perfused in situ first with EGTA solution (5.4 mM KCl, 0.44 mM KH2PO4, 140 mM NaCl, 0.34 mM Na2HPO4, 0.5 mM EGTA, 25 mM Tricine, pH 7.2)(portal vein)
灌注时间？
2. then with perfusion buffer (0.075% collagenase type I in GBSS buffer with 0.02% DNase I) 灌注时间？
3. mince and digested in digestion buffer (0.009% collagenase type I in GBSS buffer with 0.02% DNase I) at 37°C for 20-30 min.
4. The homogenate was filtered(过滤网大小? Or through gauze), centrifuged at 25g for 5 min at room temperature to remove the hepatocytes.
5. The supernatant was transferred to a new tube and centrifuged at 400g for 10 min at 4°C.
6. 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.
7. The cell fraction in the GBSS and 11.5% OptiPrep interphase was gently aspirated, mixed with GBSS, and centrifuged at 1400g for 10 min at 4°C.
8. After another wash, the final cell pellet was resuspended in RPMI 1640 medium containing penicillin(浓度?), streptomycin(浓度?), and 20% FBS, and then plated onto 24-well plates at a density of 1 × 104 cells per well in 0.5 mL culture medium or 6-well plates at a density of 1 × 105 cells per well in 1.5 mL culture medium.
Cell number and viability were assessed by the trypan blue exclusion test
这个是我找到的最像样的方法，但是中间缺少几处关键的时间和浓度等。请问有人知道吗？谢谢。

qhyu[使用道具]
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发表于 2015-10-20 11:21 资料个人空间短消息加为好友

小中大

楼主是那个医院的？我现在也准备做胰腺星状细胞的培养，请教GBSS就是盖氏平衡盐溶液是怎么配制的？多谢，多谢

ukonptp[使用道具]
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发表于 2015-10-20 11:22 资料个人空间短消息加为好友

小中大

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

ukonptp[使用道具]
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发表于 2015-10-20 11:23 资料个人空间短消息加为好友

小中大

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 (使用前加,现配)
pH 7.4
Total 1L
Digest solution
solution II containing 400 mg/l pronase and 20 mg/l DNase (使用前加,现配)
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

ukonptp[使用道具]
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发表于 2015-10-20 11:23 资料个人空间短消息加为好友

小中大

相关疾病：
46XX性发育睾丸疾病
贴一张HSCs的图，显微镜100倍下照的。图中左下和右下形状不规则的为HSC，上边的条状细胞形态上看像kupper。周末用alpha-SMA 免疫染色，确认HSCs。