g*********5
发帖数: 2533 | 1 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 |
B****n
发帖数: 22 | 2 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。。。 |
W****7
发帖数: 426 | 3 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 |
j**k
发帖数: 43 | 4 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
|
m******5
发帖数: 1383 | 5 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！
|
s******y
发帖数: 28562 | 6 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******5
发帖数: 1383 | 7 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*********5
发帖数: 2533 | |
g*********5
发帖数: 2533 | 9 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**********1
发帖数: 5204 | 10 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*******y
发帖数: 48 | 11 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******5
发帖数: 1383 | 12 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******y
发帖数: 28562 | 13 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**********1
发帖数: 5204 | 14 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*********5
发帖数: 2533 | 15 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*********5
发帖数: 2533 | 16 I would use gelatine to coat the flask. |
s******y
发帖数: 28562 | 17 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******5
发帖数: 1383 | 18 gelatin fibronectin coating is essential
【在 g*********5 的大作中提到】
: I would use gelatine to coat the flask.
|
m******5
发帖数: 1383 | 19 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*********5
发帖数: 2533 | 20 it seem AC16 is easier... |
|
|
l**********1
发帖数: 5204 | 21 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*********5
发帖数: 2533 | 22 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*********5
发帖数: 2533 | 23 can I reuse the gelatin/fibronectin solution to coat other flasks?
thanks. |
g*********5
发帖数: 2533 | 24 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*********5
发帖数: 2533 | |
l**********1
发帖数: 5204 | 26 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*********5
发帖数: 2533 | 27 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*********5
发帖数: 2533 | 28 after coating, no beat at all. |
C**S
发帖数: 522 | 29 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******5
发帖数: 1383 | 30 what is the medium you used
【在 g*********5 的大作中提到】
: after coating, no beat at all.
|
|
|
m******5
发帖数: 1383 | 31 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*********5
发帖数: 2533 | 32 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**********1
发帖数: 5204 | 33 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**********1
发帖数: 5204 | 34 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******5
发帖数: 1383 | 35 得用clayccomb medium
【在 g*********5 的大作中提到】
: sigma 的medium. 用的。
|
g*********5
发帖数: 2533 | 36 Cat 51800c from sigma.
【在 m******5 的大作中提到】
: 得用clayccomb medium
|
m******5
发帖数: 1383 | 37 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**********1
发帖数: 5204 | 38 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**********1
发帖数: 5204 | 39 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
|
