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这是我从一篇文章的Materials and Methods中copy过来的，供参考。
; q/ q/ h+ m4 K9 Z, K6 _# fCell Growth Assays
$ u. V3 f0 |1 C9 C* GGrowth curves for individual cell lines were established by! C6 Q3 B( ]- w n8 T2 g
plating a series of cultures at three different cell concentrations
0 k+ b! m4 q* w& A# T$ ^(i.e., 10^5, 3 X10^4, and 10^4 cells/mL) and calculating6 Y0 M- T8 |7 i" V- D5 n O7 Y
cell counts at 24-h intervals for 2–3 days. The population
$ c7 ^1 s2 }- {4 ]% ]) l8 \doubling time was derived by plotting cell concentration
# V6 C" E; r4 N1 J% o* oagainst time. Cell proliferation was also assessed using an8 P6 e6 E5 B/ k/ N8 y2 @' D5 n" w
enzyme-linked immunoassay to measure incorporation of
" F; w, M6 E- J5-bromo-29-deoxyuridine (BrdU) into cellular DNA
# p& q ?( I4 f8 h/ `; @ t4 S(Boehringer Mannheim). In this assay, cells (4 X10^2) were
( O2 q" m& Q V# ~# ucultured on 96-well plates and sequentially incubated with# B, r9 [; j/ s* X5 H6 }
BrdU for 2 h at regular intervals (every 8 or 12 h for 4 days),8 n5 q, Q# q$ ]7 @
fixative, and peroxidase-conjugated mouse monoclonal antibody/ `0 U: G; _3 `9 v' @
against BrdU. Peroxidase activity was detected using
2 q( {& S; m( x( @3 P w/ ?tetramethylbenzidine (TMB) as the color substrate. After
& e/ B; n. y5 x8 F7 f. zcolor development (' 15 min), absorbance was measured at# ^! V( P9 m1 b/ U- F& D/ @: ~
340 nm using a plate reader (Bio-Tech). To establish a! P, R& N7 v$ |
mitotic index for each cell line, approximately 5 X10^4 cells
4 }! Q* m" {& y, swere plated on glass coverslips, allowed to attach, and then4 K/ Z* b$ i4 s0 \: j: J: L
incubated for 12–18 h in the presence of 0.5 mCi/mL
% G# L& M) H) g2 Q4 r) @6 `[3H]thymidine (2 Ci/mmol). Following fixation of cells in
! w8 c/ p% m; x' r4 ~; p4% formalin, coverslips were coated with Kodak NTB-23 c+ L! J* h" f, Q. |! m/ E/ c+ H
emulsion, stored desiccated at 4°C for 3 days, and developed
# [% m& z0 b; ^3 G1 M* Xin D-19 developer and rapid fix (Kodak). Mitotic index
( H; z; Y% N- K7 P/ x/ gwas calculated by counting radiolabeled cells on each coverslip3 K/ ~2 V1 `) J# |$ G
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
