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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 C0 W$ i0 J: W! U0 ?8 I+ _5 T
Cell Growth Assays
W, a3 O |# E+ GGrowth curves for individual cell lines were established by4 u6 C2 \3 A) o( N7 I0 c4 |3 b. m
plating a series of cultures at three different cell concentrations
3 G3 o. p5 [! L: |: E7 ?(i.e., 10^5, 3 X10^4, and 10^4 cells/mL) and calculating6 P9 w* A7 i4 q4 y8 @: @
cell counts at 24-h intervals for 2–3 days. The population! ~/ ~& O+ R; Y4 d4 M" _0 e2 N
doubling time was derived by plotting cell concentration
8 h; }0 f c, E) c: v8 M) Bagainst time. Cell proliferation was also assessed using an( z) R8 O# ^3 r3 H6 T% T' ~! |! P
enzyme-linked immunoassay to measure incorporation of! y( W) \* |" O# V ]) m
5-bromo-29-deoxyuridine (BrdU) into cellular DNA8 x9 }' v+ ~3 _3 k% i' k8 Y
(Boehringer Mannheim). In this assay, cells (4 X10^2) were! ?' `) B2 i- R$ J8 y
cultured on 96-well plates and sequentially incubated with$ U" P4 v Q# L* |
BrdU for 2 h at regular intervals (every 8 or 12 h for 4 days),
6 L0 p6 k! _* Q nfixative, and peroxidase-conjugated mouse monoclonal antibody$ x* W4 ^1 ?4 ~1 A+ I5 r
against BrdU. Peroxidase activity was detected using3 n" T3 W' S, r6 ` o3 e
tetramethylbenzidine (TMB) as the color substrate. After3 v+ V, z( | U0 P* X( `
color development (' 15 min), absorbance was measured at9 A8 C# e+ k5 @
340 nm using a plate reader (Bio-Tech). To establish a& r0 I( F j* r' g7 S
mitotic index for each cell line, approximately 5 X10^4 cells
- Z n4 L2 |! J8 e) Qwere plated on glass coverslips, allowed to attach, and then
, c; s& O- A( y @" l6 i/ wincubated for 12–18 h in the presence of 0.5 mCi/mL
) i' x4 U3 ]/ h* e[3H]thymidine (2 Ci/mmol). Following fixation of cells in) Y1 ?* W# K5 W% m x5 Y" X
4% formalin, coverslips were coated with Kodak NTB-2
0 v# x4 n! {. l! _( s* }2 Z6 oemulsion, stored desiccated at 4°C for 3 days, and developed
4 N2 G _ a1 d h: T7 g6 p* Cin D-19 developer and rapid fix (Kodak). Mitotic index
! Y/ G( i! s& J- @. A2 K5 Fwas calculated by counting radiolabeled cells on each coverslip8 ~$ E) P1 t: N2 e2 a7 d
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
