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有人养过HL-1细胞吗？

有人养过HL-1细胞吗？# Biology - 生物学

l*d2012-06-30 07:06

1 楼

【以下文字转载自 ChuanYu 讨论区】
发信人: ninian (猪丽耶=。=), 信区: ChuanYu
标题: Re: @奔[天坑大爆料] Re: 征集帅哥美女明晚吃饭14页狂奔 (转载
发信站: BBS 未名空间站 (Fri Jul 16 20:10:29 2010, 美东)
我太八卦了。。
几个链接：
http://www.netme.cc/airways/viewthread.php?tid=536642
http://www.moko.cc/kelly/
http://blog.sina.com.cn/s/blog_48530d2b0100ehcu.html
http://webcache.googleusercontent.com/search?q=cache:oD2kMS6dMTMJ:www.tianyayidu.com/article-8441-21.html+%E6%9E%97%E8%BF%AA%E4%BD%B3&cd=29&hl=en&ct=clnk&gl=us&client=firefox-a
搜索“林”
http://citynewsweek.co

H*g2012-06-30 07:06

2 楼

这个鼠标两侧面是用光滑的硬塑料做的，太滑抓不住，所以就一直在贴东西改善，
以前贴了创可贴，但是创可贴久了以后变得很恶心，昨天在家里发现有不自行车胎的橡
胶软皮，非常好用，容易贴，时间久了也不恶心，软皮上的粗颗粒摩擦也大。
奔个图，这是以前创可贴版的。
这是现在的：
大概这么厚

g*52012-06-30 07:06

3 楼

我的细胞复苏后怎么不beat?
难道是因为flask没有coating?
请高人指点

k*e2012-06-30 07:06

4 楼

说明了什么？是不是确有一人，国色天香，无人比芳，天天品味，山高水长

【在 l******d 的大作中提到】

: 【以下文字转载自 ChuanYu 讨论区】
: 发信人: ninian (猪丽耶=。=), 信区: ChuanYu
: 标题: Re: @奔[天坑大爆料] Re: 征集帅哥美女明晚吃饭14页狂奔 (转载
: 发信站: BBS 未名空间站 (Fri Jul 16 20:10:29 2010, 美东)
: 我太八卦了。。
: 几个链接：
: http://www.netme.cc/airways/viewthread.php?tid=536642
: http://www.moko.cc/kelly/
: http://blog.sina.com.cn/s/blog_48530d2b0100ehcu.html
: http://webcache.googleusercontent.com/search?q=cache:oD2kMS6dMTMJ:www.tianyayidu.com/article-8441-21.html+%E6%9E%97%E8%BF%AA%E4%BD%B3&cd=29&hl=en&ct=clnk&gl=us&client=firefox-a

A*u2012-06-30 07:06

5 楼

就贴了个帮迪啊

【在 H********g 的大作中提到】

: 这个鼠标两侧面是用光滑的硬塑料做的，太滑抓不住，所以就一直在贴东西改善，
: 以前贴了创可贴，但是创可贴久了以后变得很恶心，昨天在家里发现有不自行车胎的橡
: 胶软皮，非常好用，容易贴，时间久了也不恶心，软皮上的粗颗粒摩擦也大。
: 奔个图，这是以前创可贴版的。
: 这是现在的：
: 大概这么厚

B*n2012-06-30 07:06

6 楼

我coat了，也没看见beating。
用的方法和试剂完全和他们文章的一样，一直怀疑。。。

g*n2012-06-30 07:06

7 楼

她也说确实在LA读书啊
谁来说说续集啥内容

g*x2012-06-30 07:06

8 楼

擦

W*72012-06-30 07:06

9 楼

最近刚要了这细胞，看来还有不少同胞再养啊。。。。
是不是试剂跟他们的不一样啊，我是连lot都跟他们定的一样的。。。
话说他的培养基也太贵鸟。。。。110多500ml

g*x2012-06-30 07:06

10 楼

擦

j*k2012-06-30 07:06

11 楼

这个细胞太难伺候了，长的又慢，每天必须换液，不换就长不好了，培养基又那么贵，
做一次试验的成本很高阿！

【在 W****7 的大作中提到】

: 最近刚要了这细胞，看来还有不少同胞再养啊。。。。
: 是不是试剂跟他们的不一样啊，我是连lot都跟他们定的一样的。。。
: 话说他的培养基也太贵鸟。。。。110多500ml

H*g2012-06-30 07:06

12 楼

以前贴邦迪呀，后来改了么

m*52012-06-30 07:06

13 楼

同养飘过，大家都用它干嘛

【在 j**k 的大作中提到】

: 这个细胞太难伺候了，长的又慢，每天必须换液，不换就长不好了，培养基又那么贵，
: 做一次试验的成本很高阿！

k*e2012-06-30 07:06

14 楼

思维不妨开拓些，贴创可贴在手上就行了

s*y2012-06-30 07:06

15 楼

听得我都怕了。
我的实验里也正准备用这个细胞西呢

【在 j**k 的大作中提到】

: 这个细胞太难伺候了，长的又慢，每天必须换液，不换就长不好了，培养基又那么贵，
: 做一次试验的成本很高阿！

m*52012-06-30 07:06

16 楼

其实没那么难养

【在 s******y 的大作中提到】

: 听得我都怕了。
: 我的实验里也正准备用这个细胞西呢

g*52012-06-30 07:06

17 楼

谢谢大家。
请教经验。

g*52012-06-30 07:06

18 楼

how about AC16?

【在 s******y 的大作中提到】

: 听得我都怕了。
: 我的实验里也正准备用这个细胞西呢

l*12012-06-30 07:06

19 楼

RE:
孵蛋的学第/妹 giantbird05 (大鸟)
Did you use Claycomb-medium ?
if not, please use it
Claycomb-medium (Sigma, Germany)
plus fibronectin/gelatine-precoated cell culture flasks.
more details about stuff protocol
pls go to
PLoS One. 2012; 7(2): e31669.
PuMeb link:
//www.ncbi.nlm.nih.gov/pmc/articles/PMC3285183/
citation:
>Cell culture
>The HL-1 cardiomyocytes (murine atrial tumor cell line) were
maintained in monolayer culture with Claycomb-medium (Sigma,
Germany), supplemented with 10% fetal calf serum (SAFCBiosciences,
USA), 1% penicillin/streptomycin, 1% glutamax
(both Invitrogen, Germany) and 1% norepinephrine (Sigma-
Aldrich, Germany) in an incubator containing 5% CO2 at a
relative humidity of 95% and 37uC. 86105 cells/cm2 were seeded
in supplemented Claycomb-medium on fibronectin/gelatine-precoated
cell culture flasks. Coating was performed using a 1 mg/ml
fibronectin-solution in 0.02% gelatine (Sigma-Aldrich, Germany)
and incubate at 37uC overnight. The Claycomb-medium, which is
specifically designed for the growth of HL-1 cells, was replaced
approximately every 48 h. Heaving reached confluence and
contractile activity, cells were maintained as subcultures using
0.05% trypsin/EDTA (Invitrogen, Germany) and subsequently
incubated under standard culture conditions.
----

【在 g*********5 的大作中提到】

: 谢谢大家。
: 请教经验。

r*y2012-06-30 07:06

20 楼

it is easy to culture HL-1, but it is some kind of difficult to maintain its
beating property. I believe it is all because of its differenciation
ability. my experience is: in addition to the use of recommemded culture
conditions and coating, changing the medium once/day is critical for its
beating, as well as its confluency. however, one of my colleagues told me
that he did exactly the same, his HL-1 was still not beating, but mine
beating well. we still could not figure out what is the problem with his HL-
1.

m*52012-06-30 07:06

21 楼

BTW, I think LZ should start from coating the flask

【在 g*********5 的大作中提到】

: 我的细胞复苏后怎么不beat?
: 难道是因为flask没有coating?
: 请高人指点

s*y2012-06-30 07:06

22 楼

没有用过，但是对此也有兴趣。

【在 g*********5 的大作中提到】

: how about AC16?

l*12012-06-30 07:06

23 楼

Here you go.
Casals G. et al. (2009)
Hypoxia induces B-type natriuretic peptide release in cell lines derived
from human cardiomyocytes.
Am J Physiol Heart Circ Physiol. 297:H550-5.
link:
//www.ncbi.nlm.nih.gov/pubmed/19542490
citation:
Cell culture.
AC16 cells were generated from primary human
ventricular cardiomyocytes, as previously described (7). This immortalized,
stable cell line can be repeatedly frozen, thawed, and propagated.
Cells were seeded (1 106 cells/well) in gelatin-coated six-well
plates and grown to confluence for 36 h in Dulbecco’s modified Eagle’s
medium (DMEM-F-12), supplemented with 12.5% fetal calf serum, 50
U/ml penicillin, and 50 g/ml streptomycin.
plus this one:
Li X. et al. (2011)
Neuronal production of transthyretin in human and murine Alzheimer's disease
J Neurosci. 31:12483-
link:
//www.ncbi.nlm.nih.gov/pubmed/21880910
发信人: sunnyday (飞鱼. 发胖中.飞不动了), 信区: Biology
标题: Re: 有人养过HL-1细胞吗？
发信站: BBS 未名空间站 (Sun Jul 1 17:15:37 2012, 美东)
没有用过，但是对此也有兴趣。

g*52012-06-30 07:06

24 楼

yes.
and use fibronectin flask.

【在 l**********1 的大作中提到】

: RE:
: 孵蛋的学第/妹 giantbird05 (大鸟)
: Did you use Claycomb-medium ?
: if not, please use it
: Claycomb-medium (Sigma, Germany)
: plus fibronectin/gelatine-precoated cell culture flasks.
: more details about stuff protocol
: pls go to
: PLoS One. 2012; 7(2): e31669.
: PuMeb link:

g*52012-06-30 07:06

25 楼

I would use gelatine to coat the flask.

s*y2012-06-30 07:06

26 楼

谢谢matrix同学！

【在 l**********1 的大作中提到】

: Here you go.
: Casals G. et al. (2009)
: Hypoxia induces B-type natriuretic peptide release in cell lines derived
: from human cardiomyocytes.
: Am J Physiol Heart Circ Physiol. 297:H550-5.
: link:
: //www.ncbi.nlm.nih.gov/pubmed/19542490
: citation:
: Cell culture.
: AC16 cells were generated from primary human

m*52012-06-30 07:06

27 楼

gelatin fibronectin coating is essential

【在 g*********5 的大作中提到】

: I would use gelatine to coat the flask.

m*52012-06-30 07:06

28 楼

it is ventricular cardiomyocye, which is more easier than atrial cell to
obtain

【在 g*********5 的大作中提到】

: how about AC16?

g*52012-06-30 07:06

29 楼

it seem AC16 is easier...

l*12012-06-30 07:06

30 楼

那是
有时侯看用途有的还非用HL 不可的只能硬着头皮上喽
for example:
while inhibition ROS release assay (possible)
can only use HL-1 not AC16
pls refer:
ROS Signalling of Inflammatory Cytokines During Trypanosoma cruzi Infection.
... discuss the potential role of ROS in chronic evolution of inflammatory
cardiovascular .... that human cardiomyocytes (AC16) produce reactive oxygen
species (ROS) ...
link:
//lib.bioinfo.pl/auid:5367921

【在 g*********5 的大作中提到】

: it seem AC16 is easier...

g*52012-06-30 07:06

31 楼

many thanks, shijie!

Infection.
inflammatory
oxygen

【在 l**********1 的大作中提到】

: 那是
: 有时侯看用途有的还非用HL 不可的只能硬着头皮上喽
: for example:
: while inhibition ROS release assay (possible)
: can only use HL-1 not AC16
: pls refer:
: ROS Signalling of Inflammatory Cytokines During Trypanosoma cruzi Infection.
: ... discuss the potential role of ROS in chronic evolution of inflammatory
: cardiovascular .... that human cardiomyocytes (AC16) produce reactive oxygen
: species (ROS) ...

g*52012-06-30 07:06

32 楼

can I reuse the gelatin/fibronectin solution to coat other flasks?
thanks.

g*52012-06-30 07:06

33 楼

if I plan do some sifxn experiment,
how many cells should I seed in 6 well plate per well?
thanks

its
HL-

【在 r*******y 的大作中提到】

: it is easy to culture HL-1, but it is some kind of difficult to maintain its
: beating property. I believe it is all because of its differenciation
: ability. my experience is: in addition to the use of recommemded culture
: conditions and coating, changing the medium once/day is critical for its
: beating, as well as its confluency. however, one of my colleagues told me
: that he did exactly the same, his HL-1 was still not beating, but mine
: beating well. we still could not figure out what is the problem with his HL-
: 1.

g*52012-06-30 07:06

34 楼

self ding

l*12012-06-30 07:06

35 楼

RE 孵蛋的学弟/妹 giantbird05 (大鸟)
of course you can do it, just Store it in freezer.
pls refer:
Gelatin/Fibronectin
• Place 0.1g gelatin into a 500 ml glass bottle. Add 500 mL of dH20
and autoclave. The
concentration of gelatin is 0.02%. Fibronectin comes as a 5 mL liquid.
Dilute 1 mL of
fibronectin in 80 mL of 0.02% gelatin in a T-75 flask. Invert to mix.
Immediately
aliquot into 8 tubes of 10 mL. Store in freezer.
Coating Flasks
• All flasks must be coated with gelatin/fibronectin the day before (
or at a minimum 2
hours before) culturing cells.
• Thaw tube of previously aliquoted gelatin/fibronectin in the water
bath, remove as
soon as it is thawed. Otherwise, fibronectin may coat the tube instead of
the flasks.
• Add 2 mL per T-25 or 6 mL per T-75. Tightly cap and place in the
incubator overnight.
Flasks can be stored in the incubator up to a couple of weeks.
• Aspirate off the liquid just before adding cells to the flasks.
from
Gilchrist, K. H., “Characterization and Validation of Cell-Based Biosensors
,” Ph.D. Thesis, Stanford University, CA, 2003.
//www.stanford.edu/group/kovacslab/cgi-bin/index.php?page=theses
and that PhD dissertation full text PDF
link is at below Google
its Top one captured
//www.google.co.uk/#hl=en&output=search&sclient=psy-ab&q=DISSERTATION++
Gilchrist%2C+K.+H++Characterization+and+Validation+of+Cell-Based+Biosensors&
oq=DISSERTATION++Gilchrist%2C+K.+H++Characterization+and+Validation+of+Cell-
Based+Biosensors&gs_l=hp.12...1054.1054.0.2534.1.1.0.0.0.0.110.110.0j1.1.0..
.0.0.4byj_QSTCbk&pbx=1&bav=on.2,or.r_gc.r_pw.r_qf.,cf.osb&fp=
d7d60ee56f904ded&biw=1138&bih=596

【在 g*********5 的大作中提到】

: can I reuse the gelatin/fibronectin solution to coat other flasks?
: thanks.

g*52012-06-30 07:06

36 楼

thanks shijie/shixiong!

【在 l**********1 的大作中提到】

: RE 孵蛋的学弟/妹 giantbird05 (大鸟)
: of course you can do it, just Store it in freezer.
: pls refer:
: Gelatin/Fibronectin
: • Place 0.1g gelatin into a 500 ml glass bottle. Add 500 mL of dH20
: and autoclave. The
: concentration of gelatin is 0.02%. Fibronectin comes as a 5 mL liquid.
: Dilute 1 mL of
: fibronectin in 80 mL of 0.02% gelatin in a T-75 flask. Invert to mix.
: Immediately

g*52012-06-30 07:06

37 楼

after coating, no beat at all.

C*S2012-06-30 07:06

38 楼

长到很密的时候才能看见几个细胞跳动。

【在 g*********5 的大作中提到】

: after coating, no beat at all.

m*52012-06-30 07:06

39 楼

what is the medium you used

【在 g*********5 的大作中提到】

: after coating, no beat at all.

m*52012-06-30 07:06

40 楼

我强烈怀疑楼主用没用claycomb medium

【在 C**S 的大作中提到】

: 长到很密的时候才能看见几个细胞跳动。

g*52012-06-30 07:06

41 楼

sigma 的medium. 用的。

【在 m******5 的大作中提到】

: 我强烈怀疑楼主用没用claycomb medium

l*12012-06-30 07:06

42 楼

LZ pls refer
above 2003 Stanford PhD dissertation
its
Chapter 5 Noise Characterization
PDF file
pp65〜80

【在 g*********5 的大作中提到】

: after coating, no beat at all.

l*12012-06-30 07:06

43 楼

hl-1 beatin rate 易受各种因子(内因or 环境因子) 影响的
具体看楼下的人家老美博士论文的相关章节

【在 g*********5 的大作中提到】

: sigma 的medium. 用的。

m*52012-06-30 07:06

44 楼

得用clayccomb medium

【在 g*********5 的大作中提到】

: sigma 的medium. 用的。

g*52012-06-30 07:06

45 楼

Cat 51800c from sigma.

【在 m******5 的大作中提到】

: 得用clayccomb medium

m*52012-06-30 07:06

46 楼

Then I have no idea, what is the purpose you are using this line?

【在 g*********5 的大作中提到】

: Cat 51800c from sigma.

l*12012-06-30 07:06

47 楼

注意media Lot的原产国
最重要的一个可能是
去甲肾上腺素
1% norepinephrine (Sigma-
Aldrich, Germany)
from
北美的有的牛来源的原料
其中的疯牛病因子 and 瘦肉精因子等比德国的EU标准高
以下的所有东西订货按原产国来订货 then try again.
Cell culture
>The HL-1 cardiomyocytes (murine atrial tumor cell line) were
maintained in monolayer culture with Claycomb-medium (Sigma,
Germany), supplemented with 10% fetal calf serum (SAFCBiosciences,
USA), 1% penicillin/streptomycin, 1% glutamax
(both Invitrogen, Germany) and 1% norepinephrine (Sigma-
Aldrich, Germany) in an incubator containing 5% CO2 at a
relative humidity of 95% and 37uC. 86105 cells/cm2 were seeded
in supplemented Claycomb-medium on fibronectin/gelatine-precoated
cell culture flasks. Coating was performed using a 1 mg/ml
fibronectin-solution in 0.02% gelatine (Sigma-Aldrich, Germany)
and incubate at 37uC overnight. The Claycomb-medium, which is
specifically designed for the growth of HL-1 cells, was replaced
approximately every 48 h. Heaving reached confluence and
contractile activity, cells were maintained as subcultures using
0.05% trypsin/EDTA (Invitrogen, Germany) and subsequently
incubated under standard culture conditions.
ps: 最后beating 了包子别忘了哈 just one= ten W-E-I B-I is ok.

【在 g*********5 的大作中提到】

: Cat 51800c from sigma.

l*12012-06-30 07:06

48 楼

Merci giantbird05 (大鸟)
寄信人: deliver (自动发信系统)
标题: 本站转帐通知单
发信站: BBS 未名空间站 (Thu Jul 12 22:19:59 2012)
来源: mitbbs.com
lotkaeuler11,您好：
giantbird05 转给您,现金(伪币): 20 .
附加留言:
thanks!
giantbird05

【在 g*********5 的大作中提到】

: 我的细胞复苏后怎么不beat?
: 难道是因为flask没有coating?
: 请高人指点