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这是我从一篇文章的Materials and Methods中copy过来的，供参考。6 A8 p' I0 \' e8 ^9 h& W/ G0 ]3 e
Cell Growth Assays
6 U8 N; _ O* e+ uGrowth curves for individual cell lines were established by+ x- o: Z a) Z' ~
plating a series of cultures at three different cell concentrations5 Y( j( F5 Z& F
(i.e., 10^5, 3 X10^4, and 10^4 cells/mL) and calculating
5 W' P9 ]0 z; m" ucell counts at 24-h intervals for 2–3 days. The population8 }2 t4 L5 W! ^9 n5 S4 t. D8 k
doubling time was derived by plotting cell concentration
4 i/ k! g- o& y h# Iagainst time. Cell proliferation was also assessed using an
. B% K# @( U7 L- D+ u1 ~enzyme-linked immunoassay to measure incorporation of2 w( j# M t. T3 ]+ ?0 g2 S. }
5-bromo-29-deoxyuridine (BrdU) into cellular DNA
! p) c1 o6 a$ s$ j2 L" C(Boehringer Mannheim). In this assay, cells (4 X10^2) were$ g* E: |$ X9 o7 e8 R
cultured on 96-well plates and sequentially incubated with& B X7 T; H m2 \3 J
BrdU for 2 h at regular intervals (every 8 or 12 h for 4 days),
- r3 q7 G9 K$ B# `6 wfixative, and peroxidase-conjugated mouse monoclonal antibody0 m! {. j) j% w9 \! P
against BrdU. Peroxidase activity was detected using
+ ?7 @% N. e/ V0 f! @! v1 g9 Stetramethylbenzidine (TMB) as the color substrate. After
+ k, N# f0 m! `& Acolor development (' 15 min), absorbance was measured at0 S# a- y1 x9 ]1 |7 q9 [
340 nm using a plate reader (Bio-Tech). To establish a" d! ~) g# i7 f- Y$ V1 V5 `
mitotic index for each cell line, approximately 5 X10^4 cells
% z" s2 t3 W, Swere plated on glass coverslips, allowed to attach, and then+ Q4 M! G+ j4 `, Q
incubated for 12–18 h in the presence of 0.5 mCi/mL
' K# P6 V- X7 P, P! Y8 F8 v[3H]thymidine (2 Ci/mmol). Following fixation of cells in
, F' L. l8 d6 U# D/ j3 g& G* F$ z5 |4% formalin, coverslips were coated with Kodak NTB-2& c- u- r4 b" @0 `! v! p
emulsion, stored desiccated at 4°C for 3 days, and developed- l" ?4 ^/ e- ~! q8 @9 u( O2 R+ H
in D-19 developer and rapid fix (Kodak). Mitotic index
( H$ p. @# s I3 }2 O/ |4 g/ C B0 Swas calculated by counting radiolabeled cells on each coverslip
. j- ]% ~4 R# ?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
