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这是我从一篇文章的Materials and Methods中copy过来的，供参考。/ a9 q# B9 ?1 K5 S
Cell Growth Assays
% o/ \/ a# R' \6 _" \Growth curves for individual cell lines were established by
& R/ u9 X. ~/ Wplating a series of cultures at three different cell concentrations
9 F. X+ [/ J& Y7 S, D6 T(i.e., 10^5, 3 X10^4, and 10^4 cells/mL) and calculating
" i7 _' b1 U* E: M$ @cell counts at 24-h intervals for 2–3 days. The population
! z" G: A' K( d l8 {. Mdoubling time was derived by plotting cell concentration
" m R! S% \( O2 B2 U! L8 t; Pagainst time. Cell proliferation was also assessed using an# \" W8 t, J+ @6 M" i
enzyme-linked immunoassay to measure incorporation of
. @5 u4 A5 s }4 Y g8 Y5-bromo-29-deoxyuridine (BrdU) into cellular DNA
) C" Z( s7 I: w3 \# U(Boehringer Mannheim). In this assay, cells (4 X10^2) were1 S8 e6 X& p5 g. u
cultured on 96-well plates and sequentially incubated with
& ?0 ?% D. c5 YBrdU for 2 h at regular intervals (every 8 or 12 h for 4 days),
4 N8 ~1 @2 W {fixative, and peroxidase-conjugated mouse monoclonal antibody
4 m1 c+ M2 ] a7 f( ^against BrdU. Peroxidase activity was detected using* u7 c, H" o6 y# W6 X
tetramethylbenzidine (TMB) as the color substrate. After
7 J: f2 X4 h* m. D0 g3 C7 xcolor development (' 15 min), absorbance was measured at1 `0 I# l6 X* s- O7 ^
340 nm using a plate reader (Bio-Tech). To establish a* ^7 I& P5 l: E6 E
mitotic index for each cell line, approximately 5 X10^4 cells5 c$ z$ v) r% a) ^8 X2 d
were plated on glass coverslips, allowed to attach, and then* S# l. K& N* s
incubated for 12–18 h in the presence of 0.5 mCi/mL
9 W( h+ ?' T$ f1 I9 l: L/ N[3H]thymidine (2 Ci/mmol). Following fixation of cells in7 E5 D. z3 D9 [( ?# z8 N, W1 z
4% formalin, coverslips were coated with Kodak NTB-2. U- Q$ L) ~& }) R2 s
emulsion, stored desiccated at 4°C for 3 days, and developed
" V3 e# Q5 z9 c! s4 p6 \in D-19 developer and rapid fix (Kodak). Mitotic index
S6 A/ Y' `) X ^: ]7 K9 ~6 w7 Gwas calculated by counting radiolabeled cells on each coverslip
+ X& i4 m; c1 I" Z1 a' Xas a percentage of total cell number. |
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发表于 2010-8-2 12:54
发表于 2010-8-2 17:08
