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。) [5 Z4 {* F& N
Cell Growth Assays  O" w4 b/ b9 o8 f
Growth curves for individual cell lines were established by! g% G- I( K" p
plating a series of cultures at three different cell concentrations# l$ {! O; p( a9 d
(i.e., 10^5, 3 X10^4, and 10^4 cells/mL) and calculating' o# ^" E  R% E% A- o% a' H. w9 |
cell counts at 24-h intervals for 2–3 days. The population" l6 f3 ^. T4 f7 s8 y8 F/ T
doubling time was derived by plotting cell concentration6 q  L9 C2 A) [+ }+ W/ i; N
against time. Cell proliferation was also assessed using an  R6 h0 r$ b& e, i
enzyme-linked immunoassay to measure incorporation of
- C2 S% e4 w- }, C5-bromo-29-deoxyuridine (BrdU) into cellular DNA: ^2 E( w# T$ C' k5 U4 B: Y- V$ y
(Boehringer Mannheim). In this assay, cells (4 X10^2) were
( \# }% }- p: d) h+ mcultured on 96-well plates and sequentially incubated with( A. K$ |3 H3 j2 _% a, B
BrdU for 2 h at regular intervals (every 8 or 12 h for 4 days),) W3 ^2 P/ b/ t- H* m& @
fixative, and peroxidase-conjugated mouse monoclonal antibody
/ j3 I, y) t% Q$ U1 Iagainst BrdU. Peroxidase activity was detected using
# C# f; `. D4 wtetramethylbenzidine (TMB) as the color substrate. After
1 F  U& r- f$ e& v* v" Bcolor development (' 15 min), absorbance was measured at+ Q" e! ?7 \6 E9 E* m
340 nm using a plate reader (Bio-Tech). To establish a- k8 T0 X7 K3 H
mitotic index for each cell line, approximately 5 X10^4 cells! B; F1 z( s: {" f" E+ H! i
were plated on glass coverslips, allowed to attach, and then
% F: c+ Z/ d$ [8 H+ |incubated for 12–18 h in the presence of 0.5 mCi/mL
( v" ?- O0 E# k: m9 m  m' L[3H]thymidine (2 Ci/mmol). Following fixation of cells in
) S) s. _7 v, z) p: O4% formalin, coverslips were coated with Kodak NTB-27 m) r8 W! h. k$ [
emulsion, stored desiccated at 4°C for 3 days, and developed9 H: S. M; h" g
in D-19 developer and rapid fix (Kodak). Mitotic index: ]" m( @+ S* Z1 O% G
was calculated by counting radiolabeled cells on each coverslip6 ?5 S: [* u& n: ]% T6 @
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 3 S3 ?) I0 h" a' H
Thanks.

hdc2001

试剂盒测的是相对数目吧，细胞计数才是绝对数目。
$ O# Q1 |$ B% A# a' r18*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 % a% V; `% K$ G, p3 q

+ n1 [( o' @1 [% p) K6 n  X  x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
( @: B/ z" |4 T4 B5 d0 W( Q
) g  h2 F* z  ]& e# Y* g
7 A+ S- @+ Y/ |" _( q/ J   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   M4 I9 U0 h, T; P9 A

2 h) j& \: r% W
6 E8 X* y0 E* ~. b0 r/ B. }    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