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这是我从一篇文章的Materials and Methods中copy过来的，供参考。
4 Q( b/ t$ L3 n' YCell Growth Assays* ? o* a2 K' Z$ c
Growth curves for individual cell lines were established by7 N2 |8 r9 s& s+ ]" N
plating a series of cultures at three different cell concentrations
1 U9 D1 z4 X! J' V$ T/ ](i.e., 10^5, 3 X10^4, and 10^4 cells/mL) and calculating- u/ T. s7 [& ^7 {- ~9 p
cell counts at 24-h intervals for 2–3 days. The population g3 [( q7 ^. O3 T4 l) j" ^
doubling time was derived by plotting cell concentration
9 Y% a2 L! Y8 N( `7 G) C2 {# Gagainst time. Cell proliferation was also assessed using an2 S- }* _2 |0 s% T$ i0 P1 S7 r
enzyme-linked immunoassay to measure incorporation of" ]$ e% N1 q U1 z, ]$ R8 j
5-bromo-29-deoxyuridine (BrdU) into cellular DNA- ~7 O1 g/ [7 B V! G% _. o" R
(Boehringer Mannheim). In this assay, cells (4 X10^2) were; T! Q/ z" v; s4 y
cultured on 96-well plates and sequentially incubated with
4 w# a, Z* g! t8 b) |- ]# p. RBrdU for 2 h at regular intervals (every 8 or 12 h for 4 days),
: a9 Q: I- L0 [$ J+ Q. b" }, g& M% Ofixative, and peroxidase-conjugated mouse monoclonal antibody4 A( Z# p: ^: R2 F: @+ g$ |) ^
against BrdU. Peroxidase activity was detected using
# Z' v( w5 ]) I) K& L& Ntetramethylbenzidine (TMB) as the color substrate. After( G* q6 k1 F8 E+ ~( N2 Y# x
color development (' 15 min), absorbance was measured at
7 X; ~" l- m0 a( H& q340 nm using a plate reader (Bio-Tech). To establish a7 @' ` v$ ?" C
mitotic index for each cell line, approximately 5 X10^4 cells
1 _+ J0 n6 g, Z' q5 g, |1 d7 awere plated on glass coverslips, allowed to attach, and then
; C& t0 V6 q, b5 oincubated for 12–18 h in the presence of 0.5 mCi/mL
7 Q3 D3 ]; w3 w( W6 h ?- _4 w[3H]thymidine (2 Ci/mmol). Following fixation of cells in
, T. k8 @2 H8 E& N h$ W X4% formalin, coverslips were coated with Kodak NTB-2) [2 i4 A' @+ K. `* b Z9 E# B
emulsion, stored desiccated at 4°C for 3 days, and developed
# R) N( Y" Z! z9 Win D-19 developer and rapid fix (Kodak). Mitotic index% A# Z i- V6 {7 G+ x z) o
was calculated by counting radiolabeled cells on each coverslip* z9 k; _' L2 `, N" J. E
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
