细胞生长曲线如何测定？

sniper

求助：细胞的生长曲线怎么测定，哪位高手提供一下详细步骤

lixiaodong

这是我从一篇文章的Materials and Methods中copy过来的，供参考。; b% ?7 b0 J% `& K( C
Cell Growth Assays
8 j9 ?8 z  i" e+ J4 _- LGrowth curves for individual cell lines were established by
+ t1 L* h7 r4 C4 e+ z) Cplating a series of cultures at three different cell concentrations7 x5 {9 O* O, @; E# b! H
(i.e., 10^5, 3 X10^4, and 10^4 cells/mL) and calculating9 ^- U, Z8 S4 N. k7 u* L( q& @
cell counts at 24-h intervals for 2–3 days. The population3 ~7 c3 D" [0 Z, ^
doubling time was derived by plotting cell concentration2 J  `) O1 H1 `+ w' n$ ]
against time. Cell proliferation was also assessed using an. T" S! O  r* h" s. q0 U* C' W
enzyme-linked immunoassay to measure incorporation of9 W- Z$ T! H6 E& c( M
5-bromo-29-deoxyuridine (BrdU) into cellular DNA
0 \7 P8 }% f8 W0 o1 k(Boehringer Mannheim). In this assay, cells (4 X10^2) were# T( ?" Y3 m9 t. j; p$ k
cultured on 96-well plates and sequentially incubated with
( n# l* l5 d* l% W2 S  t! y: \BrdU for 2 h at regular intervals (every 8 or 12 h for 4 days),  w/ u$ [0 v6 y1 R- e9 q8 i
fixative, and peroxidase-conjugated mouse monoclonal antibody) |3 ?, N4 H/ y$ F
against BrdU. Peroxidase activity was detected using; G% p+ V. @4 V1 F: R5 h
tetramethylbenzidine (TMB) as the color substrate. After& O. H4 x- V, p8 E9 {2 S
color development (' 15 min), absorbance was measured at
  ]. A: @8 n0 X$ p4 \340 nm using a plate reader (Bio-Tech). To establish a
1 d+ w6 q8 j0 S" omitotic index for each cell line, approximately 5 X10^4 cells
  B* k4 n* i! Z, ~% owere plated on glass coverslips, allowed to attach, and then
3 j) q8 G' x- R- Z7 Eincubated for 12–18 h in the presence of 0.5 mCi/mL7 `: p4 @  D& i/ X- M
[3H]thymidine (2 Ci/mmol). Following fixation of cells in
3 ~( y# f, Y9 j0 B% G4% formalin, coverslips were coated with Kodak NTB-2, u+ t* ]! _4 S. A
emulsion, stored desiccated at 4°C for 3 days, and developed
" j5 R( ?3 }* M" s/ U. ]) iin D-19 developer and rapid fix (Kodak). Mitotic index
) ~6 v! q% j7 w7 O$ `7 ywas calculated by counting radiolabeled cells on each coverslip" s8 x. N( ]4 O. n& Z
as a percentage of total cell number.

realinter

推荐碧云天产的CCK8试剂盒，MTT是上一代的产品了，用CCK8比较方便，重复性也高一些。

lixiaodong

回复 3# realinter " [" q- O$ p% j7 j" K5 @/ O
Thanks.

hdc2001

试剂盒测的是相对数目吧，细胞计数才是绝对数目。. P4 h" a1 q( X' ^" H. s" s
18*T25，三个重复，10*5个细胞接种，每天计数，取出3个T25计算平均数及活率，共测6天或7天。

sfk06

以同一密度接种后，每隔一时间断后，进行细胞计数或者通过MTT法测其吸光度值就可以了 我光这将近整了一年！唉！白费时间!

he_liang

请问以上各位大侠，是所有的细胞的铺板密度一样吗？

hljzyydx

回复 7# he_liang # t1 K) Y, ^9 B8 j
! E% v6 |8 T) |5 r* [
细胞密度的确定不取决于细胞种类，而是取决于你的实验要求，当然，大家一般会有一个习惯的铺板密度，但在不同的实验条件下应该做出调整。

he_liang

回复 8# hljzyydx : v4 g$ V2 I/ D9 {

' T7 c- q- w# x5 t+ X8 W8 J: l( d/ i3 ^4 h+ I
   嗯，好的，谢谢您！不过不知您那边的有没有什么经验可以交流一下？

hljzyydx

回复 9# he_liang . z7 I* b+ _5 d
* L- N% V* L' E

* B) s8 v7 m) L; B# Z# r3 {1 U+ t; l    没问题啊。我在这个领域也是个初学者，有什么问题我们大家可以一起探讨