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这是我从一篇文章的Materials and Methods中copy过来的，供参考。
) t/ O F$ @$ o0 l$ oCell Growth Assays* e, [; b& F9 h& u+ }7 z& R, ?
Growth curves for individual cell lines were established by
8 ?6 s! j7 K2 W& X- H, S/ ~plating a series of cultures at three different cell concentrations4 A Z& F8 O! [8 |8 {
(i.e., 10^5, 3 X10^4, and 10^4 cells/mL) and calculating+ m% [% F6 L7 v- \8 w' r
cell counts at 24-h intervals for 2–3 days. The population
9 X y( ^ j9 ] Y; N5 vdoubling time was derived by plotting cell concentration
. E. Y& l; D; g4 o) R( @. nagainst time. Cell proliferation was also assessed using an! N4 h' x* l8 g: m
enzyme-linked immunoassay to measure incorporation of
; ?& ~! f- b' p# q+ v& }5-bromo-29-deoxyuridine (BrdU) into cellular DNA
* f, M- T8 D% \1 S# B2 U: q7 a4 i(Boehringer Mannheim). In this assay, cells (4 X10^2) were4 v0 M8 C7 i+ W% @! @/ u9 @8 M" ?$ z
cultured on 96-well plates and sequentially incubated with. j# I9 |# ?/ {# i
BrdU for 2 h at regular intervals (every 8 or 12 h for 4 days),; ~/ K% |9 f6 f7 ]/ P
fixative, and peroxidase-conjugated mouse monoclonal antibody
: ~. o" O1 A: }against BrdU. Peroxidase activity was detected using* W* s% A$ E7 o% ~9 H4 r. t H
tetramethylbenzidine (TMB) as the color substrate. After- _6 I: M6 }6 X+ ~3 J( K3 E
color development (' 15 min), absorbance was measured at
/ |" ^& M: Z3 @340 nm using a plate reader (Bio-Tech). To establish a+ k& S. A9 n B5 f
mitotic index for each cell line, approximately 5 X10^4 cells0 c) C! S/ D1 M0 f2 Q' _* F1 Z
were plated on glass coverslips, allowed to attach, and then. D M8 a3 c* u+ v5 A
incubated for 12–18 h in the presence of 0.5 mCi/mL: v, y* h1 h3 A: Y
[3H]thymidine (2 Ci/mmol). Following fixation of cells in
1 h9 [% B; z2 _1 D4 w8 B" N5 t+ h4% formalin, coverslips were coated with Kodak NTB-2' P* B- c6 p8 R3 F' f; F4 j
emulsion, stored desiccated at 4°C for 3 days, and developed' V- z/ @) H3 h& R
in D-19 developer and rapid fix (Kodak). Mitotic index
2 T' a% H6 w, _9 G1 Wwas calculated by counting radiolabeled cells on each coverslip; U- |8 z: @; Q+ v( I% w) V
as 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
