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。
3 P7 [/ q8 x( V: e& MCell Growth Assays0 W1 V8 u& ^  {" I; }/ t" K* @+ Q0 n/ ~
Growth curves for individual cell lines were established by5 O2 ~( G; z) p& Y# n$ U0 w
plating a series of cultures at three different cell concentrations
1 z' N* O; U- W, q, q+ N(i.e., 10^5, 3 X10^4, and 10^4 cells/mL) and calculating
' R, g1 t  c2 O* xcell counts at 24-h intervals for 2–3 days. The population0 f  g$ Y/ {7 K; z. e6 O! k
doubling time was derived by plotting cell concentration
# r- ^3 h& H% H$ Eagainst time. Cell proliferation was also assessed using an' w; g+ ]1 a+ P0 O0 w
enzyme-linked immunoassay to measure incorporation of
+ c* o( g+ @7 M# k( {4 X5-bromo-29-deoxyuridine (BrdU) into cellular DNA
: \: M4 Z/ B, n; A5 j(Boehringer Mannheim). In this assay, cells (4 X10^2) were
: W6 r- p2 |5 ?8 y! E" _cultured on 96-well plates and sequentially incubated with
0 t5 \/ [* J4 JBrdU for 2 h at regular intervals (every 8 or 12 h for 4 days),+ t% F* U0 @% p' v4 C  a1 t. H
fixative, and peroxidase-conjugated mouse monoclonal antibody
% H6 i: P! p3 D2 y/ u* t) o3 Aagainst BrdU. Peroxidase activity was detected using% D2 m# _  ?( k- K$ Y0 i- e* }
tetramethylbenzidine (TMB) as the color substrate. After. ~, a8 h) y6 I6 f1 T% f
color development (' 15 min), absorbance was measured at' K, ^, z/ k; J- D- O
340 nm using a plate reader (Bio-Tech). To establish a
$ D" y' m1 N' f0 d2 qmitotic index for each cell line, approximately 5 X10^4 cells; m3 f  Z# @0 D( ~: W" [6 `3 Y$ Q
were plated on glass coverslips, allowed to attach, and then
9 X0 x6 z8 U: d- t# Z- `1 Cincubated for 12–18 h in the presence of 0.5 mCi/mL
. }5 _% [3 Z2 E6 q8 {* a[3H]thymidine (2 Ci/mmol). Following fixation of cells in, G% O3 B- q6 F9 F
4% formalin, coverslips were coated with Kodak NTB-2# B/ ~6 ^# [9 o5 f% W) ~
emulsion, stored desiccated at 4°C for 3 days, and developed* m" J' H$ L* I6 A
in D-19 developer and rapid fix (Kodak). Mitotic index
$ h; m* h8 q( A9 N& M9 @was calculated by counting radiolabeled cells on each coverslip
' |* I$ K* d) D$ J* N. d  Q  bas 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 4 m) ]2 x; P' y# P, g# }
Thanks.

hdc2001

试剂盒测的是相对数目吧，细胞计数才是绝对数目。
9 M1 N/ M' f/ J6 }& J* n18*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 5 W/ ~2 Z; ]! t- f! z  y

; N4 X6 M) i0 e: y! S2 J2 I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
! A! M& U- T& a; m4 g2 f$ n$ d8 R5 ]' {+ @& g

3 J3 n) j0 R( ]4 ?( n1 Z   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 ) C$ [5 L3 ~: A$ c3 Y& p

4 }+ s/ n# O7 T% H
) }) ^( q% Q6 @3 K5 U2 g9 @% a    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