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这是我从一篇文章的Materials and Methods中copy过来的，供参考。
; a+ H4 `5 r) yCell Growth Assays
1 ?6 G+ f6 N D1 e4 SGrowth curves for individual cell lines were established by. r V& a7 l: q, ]4 ~! h
plating a series of cultures at three different cell concentrations& d5 _# m2 U0 E7 x( |. D
(i.e., 10^5, 3 X10^4, and 10^4 cells/mL) and calculating# j" T* o p) B( I5 L
cell counts at 24-h intervals for 2–3 days. The population
6 D: I Q3 L$ k# tdoubling time was derived by plotting cell concentration u( ^: D2 Z* ^0 k8 r) Z) T
against time. Cell proliferation was also assessed using an- U) C/ p9 }0 q% `
enzyme-linked immunoassay to measure incorporation of! ]& y; j( g+ f$ a( Q
5-bromo-29-deoxyuridine (BrdU) into cellular DNA2 l; {2 `$ D# M' V; Z. ^
(Boehringer Mannheim). In this assay, cells (4 X10^2) were- }. v6 h7 \0 j Q3 S+ e5 g
cultured on 96-well plates and sequentially incubated with" B7 G- o+ H8 u: e6 |3 d0 W8 ^$ d) J4 h" Z
BrdU for 2 h at regular intervals (every 8 or 12 h for 4 days),
" w7 c$ Y7 g5 |7 P8 u4 {+ tfixative, and peroxidase-conjugated mouse monoclonal antibody4 Y: n. [4 K6 x7 M7 b) p
against BrdU. Peroxidase activity was detected using# w. Q7 v3 g+ a+ ~' ?: f
tetramethylbenzidine (TMB) as the color substrate. After
0 z8 f/ z- m# |' s4 ]' m, v; @3 Q& K% t; Ecolor development (' 15 min), absorbance was measured at+ u' A; R4 ~ ^
340 nm using a plate reader (Bio-Tech). To establish a
: Y0 C" T% o& x9 B+ Pmitotic index for each cell line, approximately 5 X10^4 cells" _% x6 |$ h( f0 E* X" N) s. _* Z% j
were plated on glass coverslips, allowed to attach, and then5 A, v" Q2 O, {
incubated for 12–18 h in the presence of 0.5 mCi/mL% `+ Y ^- C7 F2 P- r8 L& i4 ]
[3H]thymidine (2 Ci/mmol). Following fixation of cells in- @! P8 F$ Q+ u
4% formalin, coverslips were coated with Kodak NTB-2
- U+ e# S: o; @7 P( c! `" i, G5 P. gemulsion, stored desiccated at 4°C for 3 days, and developed
v/ B# j9 n1 x2 ]( `* b8 |in D-19 developer and rapid fix (Kodak). Mitotic index
! P9 P/ X) ]2 f( l: ywas calculated by counting radiolabeled cells on each coverslip
9 g. N/ q- i. S4 v# \4 q) `/ 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
