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这是我从一篇文章的Materials and Methods中copy过来的，供参考。* [! \6 Y* N) ^9 q* S
Cell Growth Assays! }, t2 K. _" F
Growth curves for individual cell lines were established by
# z2 z& I8 N; y) i* M' d0 g; Nplating a series of cultures at three different cell concentrations
; g% _/ Z5 G2 D l X(i.e., 10^5, 3 X10^4, and 10^4 cells/mL) and calculating. O- g& |& b0 q e' J. d
cell counts at 24-h intervals for 2–3 days. The population
- t" R9 F' S9 z. fdoubling time was derived by plotting cell concentration& k9 l; S6 ?! Y) f( o
against time. Cell proliferation was also assessed using an5 z$ e9 I* M$ v+ O/ J
enzyme-linked immunoassay to measure incorporation of; T6 x+ g5 N2 _! L
5-bromo-29-deoxyuridine (BrdU) into cellular DNA
4 v- o1 h/ ^5 n& z(Boehringer Mannheim). In this assay, cells (4 X10^2) were
& p# G8 E8 u) q% Z3 ccultured on 96-well plates and sequentially incubated with5 Y B7 |% t' C
BrdU for 2 h at regular intervals (every 8 or 12 h for 4 days),4 D8 @# [8 n p' L6 U6 K# u
fixative, and peroxidase-conjugated mouse monoclonal antibody F3 U: x( |8 x2 {. y% t1 U
against BrdU. Peroxidase activity was detected using, ^. [# h/ z5 p5 g3 k' ?6 b Y9 z
tetramethylbenzidine (TMB) as the color substrate. After
7 Y1 p# C( H' c, ^color development (' 15 min), absorbance was measured at
# V* Q5 r! g$ D; \, m" N2 B/ y' j4 q340 nm using a plate reader (Bio-Tech). To establish a0 r$ R' n# T" e3 m+ ]& ~
mitotic index for each cell line, approximately 5 X10^4 cells
( d: D: q' C2 I" r3 z2 uwere plated on glass coverslips, allowed to attach, and then- O4 V2 b2 r0 ~+ y) w( }0 T
incubated for 12–18 h in the presence of 0.5 mCi/mL
9 h7 N+ d0 {7 {* V- D o) k* a[3H]thymidine (2 Ci/mmol). Following fixation of cells in9 j/ M8 J7 f/ X, @
4% formalin, coverslips were coated with Kodak NTB-2
* @: f" W( E4 N! V6 ]0 n# Lemulsion, stored desiccated at 4°C for 3 days, and developed
0 k! T1 d7 q' Bin D-19 developer and rapid fix (Kodak). Mitotic index
, j* ?. x3 i iwas calculated by counting radiolabeled cells on each coverslip
6 N) E, Y5 t* I3 c% vas a percentage of total cell number. |
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发表于 2010-9-13 09:33
发表于 2010-8-2 17:08
