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这是我从一篇文章的Materials and Methods中copy过来的，供参考。* v. k; |1 ]! v, L- \
Cell Growth Assays
3 z. d2 M) C+ F, H- fGrowth curves for individual cell lines were established by
1 }3 O: O# {+ `7 Cplating a series of cultures at three different cell concentrations
$ x/ b% o6 a+ O# H(i.e., 10^5, 3 X10^4, and 10^4 cells/mL) and calculating4 P, G/ q5 ^# D
cell counts at 24-h intervals for 2–3 days. The population& i' @/ F6 U; z2 O; l1 O q
doubling time was derived by plotting cell concentration$ x/ ~4 a; M* \% x: Z6 z
against time. Cell proliferation was also assessed using an; t+ M' n' J2 } e# D
enzyme-linked immunoassay to measure incorporation of
+ d7 j( U" O' k; q/ e5-bromo-29-deoxyuridine (BrdU) into cellular DNA0 b. u. ]4 w0 Y0 ?% f( `0 `# O
(Boehringer Mannheim). In this assay, cells (4 X10^2) were
! a5 [) U& v& [8 zcultured on 96-well plates and sequentially incubated with5 m* V4 Z9 {. v" C8 M! x& ?
BrdU for 2 h at regular intervals (every 8 or 12 h for 4 days),
5 M) b( {) Z1 o) e- d- Lfixative, and peroxidase-conjugated mouse monoclonal antibody, X* u' W5 Y& N7 j" H
against BrdU. Peroxidase activity was detected using
8 E! B' }% e4 H; m* z- Xtetramethylbenzidine (TMB) as the color substrate. After
: T* x. g" w* A; Y7 Kcolor development (' 15 min), absorbance was measured at
0 e% X2 T& u! A, [& Y j+ p340 nm using a plate reader (Bio-Tech). To establish a
; @: o1 K, X2 s7 w# A7 j# P Lmitotic index for each cell line, approximately 5 X10^4 cells
6 a) T E0 j: ?7 B+ fwere plated on glass coverslips, allowed to attach, and then
- ^* r2 |: s+ b+ f% y+ a+ Nincubated for 12–18 h in the presence of 0.5 mCi/mL
6 D8 w9 \, ~8 X% D7 u# F; ~: ^[3H]thymidine (2 Ci/mmol). Following fixation of cells in% ?' I* H& f% q8 A, ` t
4% formalin, coverslips were coated with Kodak NTB-20 D" S" [; n+ e, q
emulsion, stored desiccated at 4°C for 3 days, and developed3 |6 k# U% ]$ R4 }
in D-19 developer and rapid fix (Kodak). Mitotic index/ B6 d) i* ?: W/ R/ x/ G
was calculated by counting radiolabeled cells on each coverslip4 r9 N$ z( [$ X4 ?
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
