p**i
发帖数: 3525 | 1 最近总苦于换medium时细胞被冲起来，多小心都不行。
可以提高coat时的gelatin的浓度吗?
谢谢 |
E**O
发帖数: 62 | |
p**i
发帖数: 3525 | 3 我需要细胞密度很高
【在 E**O 的大作中提到】
: 细胞密度低一点！细胞不喜欢很拥挤！
|
s******e
发帖数: 163 | |
p**i
发帖数: 3525 | 5 zkss
【在 s******e 的大作中提到】
: 往外倒
|
h********n
发帖数: 4079 | |
p**i
发帖数: 3525 | 7 Many thanks. Is it stronger than gelatin?
Collagan I or IV?
3T3 cells 最适合用什么呢?
【在 h********n 的大作中提到】
: collagen coated dish
|
h********n
发帖数: 4079 | 8 具体哪个细胞用那个coating的dish我不知道, 要靠自己试.
BTW, 3T3我没养过. |
s******y
发帖数: 28562 | 9 Our NIH 3T3 cells are very well adhesive even without any coating.
If yours don't still to the wall tight enough, very likely they are
contaminated with mycoplasma
【在 p**i 的大作中提到】
: 最近总苦于换medium时细胞被冲起来，多小心都不行。
: 可以提高coat时的gelatin的浓度吗?
: 谢谢
|
p**i
发帖数: 3525 | 10 平时还是很贴壁的，问题在到了confluence之后。
有什么简便的方法检测和去除mycoplasma?
【在 s******y 的大作中提到】
: Our NIH 3T3 cells are very well adhesive even without any coating.
: If yours don't still to the wall tight enough, very likely they are
: contaminated with mycoplasma
|
|
|
N******o
发帖数: 3053 | 11 如果你需要细胞密度很高做实验的话，可以先多个Plate养，然后收集起来加到
RetroNectin（Takarabio，Japan)修饰Plate上面。
RetroNectin个破东西超级厉害，当然也是超级贵，你只有用高浓度Trypsin才能剥离细
胞，而且剥离后一定加入PBS冲开细胞，如果你加入培养液，里面的血清中和Trypsin以
后细胞又被RetroNectin粘上去了。
【在 p**i 的大作中提到】
: 我需要细胞密度很高
|
b********i
发帖数: 73 | 12 Hi, there: May I have a question about 3T3-L1 preadipocyte differentation?
In induction medium, I will use
10% FBS, 5ug/ml insulin, 0.5mM IBMX and 1uM DEX for 4 days. Then keep in
5ug/ml insulin for another 4
days. Any difference from your protocol?
【在 s******y 的大作中提到】
: Our NIH 3T3 cells are very well adhesive even without any coating.
: If yours don't still to the wall tight enough, very likely they are
: contaminated with mycoplasma
|
s******y
发帖数: 28562 | 13 如果confluent 就不贴壁的话，有两个可能：
1。mycoplasma contamination
2. cell type transformation
我们是用PCR-based method, 非常简单，
很多公司有卖的，大概就叫 mycoplasma detection kit
没有好办法去除，发现被污染就就整个细胞株都扔了重新在可靠来源买一个。
我当时用这个3T3的时候，试了学校里几个实验室来源的细胞都有污染，
后来干脆从ATCC买了一个新的，反正又不贵
【在 p**i 的大作中提到】
: 平时还是很贴壁的，问题在到了confluence之后。
: 有什么简便的方法检测和去除mycoplasma?
|
s******y
发帖数: 28562 | 14 不好意思我们不是做这个的，我对这个实验不了解
?
【在 b********i 的大作中提到】
: Hi, there: May I have a question about 3T3-L1 preadipocyte differentation?
: In induction medium, I will use
: 10% FBS, 5ug/ml insulin, 0.5mM IBMX and 1uM DEX for 4 days. Then keep in
: 5ug/ml insulin for another 4
: days. Any difference from your protocol?
|
p**i
发帖数: 3525 | 15 谢谢，贵就算了。我老板反复教导我们要用最省钱的方式做实验。
【在 N******o 的大作中提到】
: 如果你需要细胞密度很高做实验的话，可以先多个Plate养，然后收集起来加到
: RetroNectin（Takarabio，Japan)修饰Plate上面。
: RetroNectin个破东西超级厉害，当然也是超级贵，你只有用高浓度Trypsin才能剥离细
: 胞，而且剥离后一定加入PBS冲开细胞，如果你加入培养液，里面的血清中和Trypsin以
: 后细胞又被RetroNectin粘上去了。
|
p**i
发帖数: 3525 | 16 这样啊。可是这些我做的stable lines。。。
【在 s******y 的大作中提到】
: 如果confluent 就不贴壁的话，有两个可能：
: 1。mycoplasma contamination
: 2. cell type transformation
: 我们是用PCR-based method, 非常简单，
: 很多公司有卖的，大概就叫 mycoplasma detection kit
: 没有好办法去除，发现被污染就就整个细胞株都扔了重新在可靠来源买一个。
: 我当时用这个3T3的时候，试了学校里几个实验室来源的细胞都有污染，
: 后来干脆从ATCC买了一个新的，反正又不贵
|
n********k
发帖数: 2818 | 17 must be a newbie and a bit careless researcher:))) this 3t3 is not that 3t3.
..
?
【在 b********i 的大作中提到】
: Hi, there: May I have a question about 3T3-L1 preadipocyte differentation?
: In induction medium, I will use
: 10% FBS, 5ug/ml insulin, 0.5mM IBMX and 1uM DEX for 4 days. Then keep in
: 5ug/ml insulin for another 4
: days. Any difference from your protocol?
|
n********k
发帖数: 2818 | 18 u can use drug to treat them to get rid of the containimation if u have no
choice..otherwise just start all over...additionally, it could be the
manufacture plates/gelitin changed...I had a big struggle before my
graduation due to a manufacture change from BD, but they never admit the
problem even if I had stone-solid evidence...they did send me several case
of plates free but they never figured out the problem...later on, I found
out online even their own researchers was reporting/asking about
【在 p**i 的大作中提到】
: 这样啊。可是这些我做的stable lines。。。
|
p**i
发帖数: 3525 | 19 haha, thanks
I will at least cite:
neverthink, sunnyday, et al, mitbbs, 2010, ....
【在 n********k 的大作中提到】
: u can use drug to treat them to get rid of the containimation if u have no
: choice..otherwise just start all over...additionally, it could be the
: manufacture plates/gelitin changed...I had a big struggle before my
: graduation due to a manufacture change from BD, but they never admit the
: problem even if I had stone-solid evidence...they did send me several case
: of plates free but they never figured out the problem...later on, I found
: out online even their own researchers was reporting/asking about
|
