l**x
发帖数: 52 | 1 现在用的是C2C12细胞，经常一不小心就长满整个平板，ATCC说这个细胞株不能长满平
板，另外，这个细胞表达myosin regulatory light chain的磷酸化特别低，很难用
WESTERN测出来,用的是Cell Signaling的regulatory light chain S19的磷酸化抗体。
大家有什么好的细胞株推荐吗？特别是来自于skeletal muscle的。另外，对抗体有什
么推荐的最好了，谢谢 |
n********s
发帖数: 1367 | 2 这个细胞不能长满是因为会分化，我奇怪你做myosin难道不需要分化myotube吗？
myosin的表达量在细胞里应该很低，但是在myotube里应该很高
★ Sent from iPhone App: iReader Mitbbs Lite 7.28 |
n********k
发帖数: 2818 | 3 C2C12 is both a very easy and very hard line to work with and I am not aware
any other better established line other than this one or one could go with
primary lines...
Anyhow, If one follows closely certain routines, this line is very easy--
-passage before 80-90% confluence with no local overgrowth...Overgrowth is a
huge problem with this line, after 3-5 times, the entire culture will be
changed...Also, the confluence etc matters a lot for differentiation...
In addition, there are at least two different C2c12 lines out there if not
three, ATCC one is very prone to differentiation and harder to keep while
the other one from is easier...they look and behave the same for non-experts
but so different for experts...both will do the job but one has to know it
before one could get anything meaningful out of the lines..
For a good reference, Check a protocol Chapter in "Cell Death in Myoblasts
and Muscles", Lawrence M Schwartz etc; in the note session, there are quite
a bit little tricks as how to deal with C2C12 cells...the key is to be very
attentive: let cells control your schedule, rather than the other way around...
【在 l**x 的大作中提到】
: 现在用的是C2C12细胞，经常一不小心就长满整个平板，ATCC说这个细胞株不能长满平
: 板，另外，这个细胞表达myosin regulatory light chain的磷酸化特别低，很难用
: WESTERN测出来,用的是Cell Signaling的regulatory light chain S19的磷酸化抗体。
: 大家有什么好的细胞株推荐吗？特别是来自于skeletal muscle的。另外，对抗体有什
: 么推荐的最好了，谢谢
|
w***e
发帖数: 269 | 4 I like L6 line better than C2C12. You can try that as well. |
n********k
发帖数: 2818 | 5 Are there many genomic level data available for L6 line? Maybe it has been
changing in the past 5-10 ys... I thought L6 was not nearly commonly used as
C2 lines...
【在 w***e 的大作中提到】
: I like L6 line better than C2C12. You can try that as well.
|
l**x
发帖数: 52 | 6 你说的对。目前的实验暂时不需要分化，看到的文献里也没有分化。cell signaling的
western结果显示regulatory light chain磷酸化水平非常好，我怀疑他们刺激细胞使
磷酸化水平增高，但是technical support说没有刺激，而且用的是非分化的细胞。已
经被这个试验折磨一两个月了，头都晕了
【在 n********s 的大作中提到】
: 这个细胞不能长满是因为会分化，我奇怪你做myosin难道不需要分化myotube吗？
: myosin的表达量在细胞里应该很低，但是在myotube里应该很高
: ★ Sent from iPhone App: iReader Mitbbs Lite 7.28
|
l**x
发帖数: 52 | 7 谢谢neverthink。你给的信息很全，很有用。你知道，可以从哪买到另一种C2C12吗？
另外，目前用这个细胞做siRNA实验也不是特别成功，siRNA从Dharmacon买的，应该质
量可靠，试了Qiagen hiperfect和Invitron lipofectamine RNAiMAX，效果都不怎么好
. 用过Qiagen hiperfect做过别的几株细胞，knockdown都很好。不知道是C2C12的原因
还是怎么的
aware
with
【在 n********k 的大作中提到】
: C2C12 is both a very easy and very hard line to work with and I am not aware
: any other better established line other than this one or one could go with
: primary lines...
: Anyhow, If one follows closely certain routines, this line is very easy--
: -passage before 80-90% confluence with no local overgrowth...Overgrowth is a
: huge problem with this line, after 3-5 times, the entire culture will be
: changed...Also, the confluence etc matters a lot for differentiation...
: In addition, there are at least two different C2c12 lines out there if not
: three, ATCC one is very prone to differentiation and harder to keep while
: the other one from is easier...they look and behave the same for non-experts
|
l**x
发帖数: 52 | 8 谢谢，准备问问看周边谁有没有这个细胞株，弄点来试试。
【在 w***e 的大作中提到】
: I like L6 line better than C2C12. You can try that as well.
|
y****i
发帖数: 2194 | 9 primary myoblast is not that difficult to make and way better than C2C12.
【在 l**x 的大作中提到】
: 现在用的是C2C12细胞，经常一不小心就长满整个平板，ATCC说这个细胞株不能长满平
: 板，另外，这个细胞表达myosin regulatory light chain的磷酸化特别低，很难用
: WESTERN测出来,用的是Cell Signaling的regulatory light chain S19的磷酸化抗体。
: 大家有什么好的细胞株推荐吗？特别是来自于skeletal muscle的。另外，对抗体有什
: 么推荐的最好了，谢谢
|
n********k
发帖数: 2818 | 10 second this...that said, the LZ seems a newbie too and so established line
could have some advantage...
For C2c12 lines, one could just request from different labs around, I forget
whether the other line is from helen Blau's lab at stanford or not...they
have a c2 line which is even tricker...
For knock-down, I never tried in the line and I don't see the reason but I
guess u could try the same shRNAs in c2 together with some other line, see
what happens...
【在 y****i 的大作中提到】
: primary myoblast is not that difficult to make and way better than C2C12.
|
|
|
b***2
发帖数: 348 | 11 这么恐怖？我前几天还和Schwartz讨论过这个细胞。他亲口告诉我，这个细胞很好搞定
，10% FBS的DMEM随便养养就好了。如果想分化，就换成2%的Horse serum。如果换回10
%FBS，细胞又全部回到stem cell状态了。
aware
with
【在 n********k 的大作中提到】
: C2C12 is both a very easy and very hard line to work with and I am not aware
: any other better established line other than this one or one could go with
: primary lines...
: Anyhow, If one follows closely certain routines, this line is very easy--
: -passage before 80-90% confluence with no local overgrowth...Overgrowth is a
: huge problem with this line, after 3-5 times, the entire culture will be
: changed...Also, the confluence etc matters a lot for differentiation...
: In addition, there are at least two different C2c12 lines out there if not
: three, ATCC one is very prone to differentiation and harder to keep while
: the other one from is easier...they look and behave the same for non-experts
|
n********k
发帖数: 2818 | 12 Well, did I say it is both very easy and very hard...frankly, this is a
problem with many stem cell-like lines...I believe Dr. Schwartz was just
trying to be very positive/encouraging...
10
【在 b***2 的大作中提到】
: 这么恐怖？我前几天还和Schwartz讨论过这个细胞。他亲口告诉我，这个细胞很好搞定
: ，10% FBS的DMEM随便养养就好了。如果想分化，就换成2%的Horse serum。如果换回10
: %FBS，细胞又全部回到stem cell状态了。
:
: aware
: with
|
n********k
发帖数: 2818 | 13 不过，C2C12确实是一个heterogeneous population，即时low serum诱导分化，也总有
一小部分的细胞是不分化的。把这部分细胞拿出来，可以继续长。
one can repeat this several times, and then no cells can differentiate
anymore...another thing is even if one makes clones from the line, it would
behave the same...there are indeed something rather interesting here and
c2c12 has been so very useful in the field...if I have extra hands and funds
, I may do something with the lines besides my main direction in neural
stuff...have any interest to collaborate on this?
的， |
n********k
发帖数: 2818 | 14 Yes, 20% FBS (5% FBS+15%BCS or hyclone works well too and much cheaper) will
do better...but it is a bit costly and doesn't really get rid of the
problem though...can play with the co2 level too...
的， |
g*********5
发帖数: 2533 | 15 siRNA work not good because it is a mice cell line and hard for transfection.
【在 l**x 的大作中提到】
: 谢谢neverthink。你给的信息很全，很有用。你知道，可以从哪买到另一种C2C12吗？
: 另外，目前用这个细胞做siRNA实验也不是特别成功，siRNA从Dharmacon买的，应该质
: 量可靠，试了Qiagen hiperfect和Invitron lipofectamine RNAiMAX，效果都不怎么好
: . 用过Qiagen hiperfect做过别的几株细胞，knockdown都很好。不知道是C2C12的原因
: 还是怎么的
:
: aware
: with
|
g*********5
发帖数: 2533 | 16 I passage c2c12 cells when confluence...
no confluence passage is it important for the further study?
thanks
【在 l**x 的大作中提到】
: 现在用的是C2C12细胞，经常一不小心就长满整个平板，ATCC说这个细胞株不能长满平
: 板，另外，这个细胞表达myosin regulatory light chain的磷酸化特别低，很难用
: WESTERN测出来,用的是Cell Signaling的regulatory light chain S19的磷酸化抗体。
: 大家有什么好的细胞株推荐吗？特别是来自于skeletal muscle的。另外，对抗体有什
: 么推荐的最好了，谢谢
|
g*********5
发帖数: 2533 | 17 I tried shRNA, Very good.
shRNA from openbiosystem.
forget
【在 n********k 的大作中提到】
: second this...that said, the LZ seems a newbie too and so established line
: could have some advantage...
: For C2c12 lines, one could just request from different labs around, I forget
: whether the other line is from helen Blau's lab at stanford or not...they
: have a c2 line which is even tricker...
: For knock-down, I never tried in the line and I don't see the reason but I
: guess u could try the same shRNAs in c2 together with some other line, see
: what happens...
|
g*********5
发帖数: 2533 | 18 总有一小部分的细胞是不分化的。把这部分细胞拿出来，
可以继续长. yes.
I use 10% FBS.
的， |
n********k
发帖数: 2818 | 19 depends on the definition of confluence, it could mean different things to
different people...When I was training people, we would work side by side on
this...repeatedly over confluence would be a big concern for the line's
differentiation property and thus experimental consistence...When I was
working in the field, unless it is a night and day difference, before I
would ever believe my results concerning differentiation phenotypes, I would
have tried at least 2 different batches of cells and 3 different confluence
for differentiation...This line can be shifting as culturing and the
differentiation varies quite a bit with the cell density at the time of
induction...
【在 g*********5 的大作中提到】
: I passage c2c12 cells when confluence...
: no confluence passage is it important for the further study?
: thanks
|
l**x
发帖数: 52 | 20 不知道原来siRNA对老鼠的细胞株效果不好
transfection.
【在 g*********5 的大作中提到】
: siRNA work not good because it is a mice cell line and hard for transfection.
|
|
|
f*****f
发帖数: 195 | 21 不是siRNA对老鼠细胞株效果不好，而是因为C2C12或primary myoblast转染效率偏低，
但也有人转染siRNA成功了的。
可以用病毒转染miRNA质粒，
或者脂质体转染后加质粒抗性标记筛选（如puromycin等）效果也很好。
【在 l**x 的大作中提到】
: 不知道原来siRNA对老鼠的细胞株效果不好
:
: transfection.
|
n********k
发帖数: 2818 | 22 I think C2c12 transfection efficiency is not that bad at all for functional
studies...one just has to be know the right assay/approach to determine the
efficiency of the knockdown...or figure out a protocol to do highly
efficiently siRNA transfection...in general, siRNA is a lot easier to be
transfected compare to plasmids...
【在 f*****f 的大作中提到】
: 不是siRNA对老鼠细胞株效果不好，而是因为C2C12或primary myoblast转染效率偏低，
: 但也有人转染siRNA成功了的。
: 可以用病毒转染miRNA质粒，
: 或者脂质体转染后加质粒抗性标记筛选（如puromycin等）效果也很好。
|
b****s
发帖数: 148 | 23 nah..
once C2C12 differentiated, it will never re-enter cell cycle.
2% horse serum is good for inducing differentiation, but switching back to
higher % serum won't help growth anymore if myogenesis is in progress.
【在 n********k 的大作中提到】
: Well, did I say it is both very easy and very hard...frankly, this is a
: problem with many stem cell-like lines...I believe Dr. Schwartz was just
: trying to be very positive/encouraging...
:
: 10
|
b****s
发帖数: 148 | 24 C2C12 needs to be passaged when under 40-50% conflu.
over 60% confluency will potentially lead to partial differentiation.. and
windup with less viable cells.
【在 g*********5 的大作中提到】
: I passage c2c12 cells when confluence...
: no confluence passage is it important for the further study?
: thanks
|
