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这是我从一篇文章的Materials and Methods中copy过来的，供参考。
' s; ], g5 o9 G! Q" qCell Growth Assays
i8 N* z: k7 C" A/ _) tGrowth curves for individual cell lines were established by
+ I) ?4 B' g. A# y2 W% kplating a series of cultures at three different cell concentrations
* y- }8 a$ \+ Y% H8 u7 s(i.e., 10^5, 3 X10^4, and 10^4 cells/mL) and calculating
6 S7 A) T2 \2 k+ ]cell counts at 24-h intervals for 2–3 days. The population
+ @4 c7 C, ]5 H/ Tdoubling time was derived by plotting cell concentration
$ B& Y1 n- B" \1 h+ r6 ?; E7 Uagainst time. Cell proliferation was also assessed using an
; G- S, N% g( E3 s- B8 b: Cenzyme-linked immunoassay to measure incorporation of8 }8 @5 Q8 }: W4 T H
5-bromo-29-deoxyuridine (BrdU) into cellular DNA
' u6 ^6 Y a+ ?3 ^(Boehringer Mannheim). In this assay, cells (4 X10^2) were+ [3 p8 x3 \) T
cultured on 96-well plates and sequentially incubated with
( { q% ]3 g+ D6 g8 V3 j9 @BrdU for 2 h at regular intervals (every 8 or 12 h for 4 days),
R+ k& K0 {& Q7 @% pfixative, and peroxidase-conjugated mouse monoclonal antibody* [6 y: D; n* m% a' \! i
against BrdU. Peroxidase activity was detected using
5 r9 T& a7 b$ k+ t1 C) ~7 e4 H5 O7 Otetramethylbenzidine (TMB) as the color substrate. After
% U8 j" l( m: j! Fcolor development (' 15 min), absorbance was measured at7 a) d- ]1 l$ X$ h; H8 U
340 nm using a plate reader (Bio-Tech). To establish a
4 H: _. j1 K5 ?mitotic index for each cell line, approximately 5 X10^4 cells
1 O( |% O5 |. H# h% o' x: N% ]9 Dwere plated on glass coverslips, allowed to attach, and then9 K: E5 w' g# A, o
incubated for 12–18 h in the presence of 0.5 mCi/mL
/ Y/ w* F5 W2 Q[3H]thymidine (2 Ci/mmol). Following fixation of cells in
" P! O% W8 H( L4% formalin, coverslips were coated with Kodak NTB-2
9 E! j6 R5 v: Oemulsion, stored desiccated at 4°C for 3 days, and developed
+ `. k! o W: B4 F9 h pin D-19 developer and rapid fix (Kodak). Mitotic index
1 `, @6 I2 e/ Q. Q, \ fwas calculated by counting radiolabeled cells on each coverslip: v: ?0 k4 J6 C5 e1 T% G, x) T& C
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
