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这是我从一篇文章的Materials and Methods中copy过来的，供参考。
: y3 f" z7 ?; ^+ ^* t" h |Cell Growth Assays
4 ?1 @2 {: z* k+ E( W, {( vGrowth curves for individual cell lines were established by9 S( M, c e! i" j! k, w( I! D
plating a series of cultures at three different cell concentrations
5 [: ^ U* F$ |& ^% Y; V(i.e., 10^5, 3 X10^4, and 10^4 cells/mL) and calculating5 |0 W3 P5 h% ~# f% A6 I% }6 r
cell counts at 24-h intervals for 2–3 days. The population
" ^/ z" }) u F9 c' Ndoubling time was derived by plotting cell concentration2 A; c; L0 Y2 d
against time. Cell proliferation was also assessed using an
/ K7 j2 Q/ Y O- c. `& G, X$ lenzyme-linked immunoassay to measure incorporation of
* F; H" r- a; Z0 v5-bromo-29-deoxyuridine (BrdU) into cellular DNA5 k8 A2 a3 t, F3 a9 K
(Boehringer Mannheim). In this assay, cells (4 X10^2) were+ C0 M/ ?& |6 r" {1 g
cultured on 96-well plates and sequentially incubated with
K% G) K+ p2 Q9 b _BrdU for 2 h at regular intervals (every 8 or 12 h for 4 days),+ R0 G" K# i( G1 e ~8 i- K
fixative, and peroxidase-conjugated mouse monoclonal antibody
. H( q0 R, I# l, b1 wagainst BrdU. Peroxidase activity was detected using- Z3 X) P' t; c# O' `; p6 E) z
tetramethylbenzidine (TMB) as the color substrate. After& Y: L$ o; x- V& A
color development (' 15 min), absorbance was measured at
! {8 U; J6 j1 X( e340 nm using a plate reader (Bio-Tech). To establish a
" u+ Y; [4 A) O" ]4 fmitotic index for each cell line, approximately 5 X10^4 cells
* }! z; i/ B* M9 o- X% b9 M5 swere plated on glass coverslips, allowed to attach, and then# e+ R! J* e/ O I( t" z/ k1 @- H
incubated for 12–18 h in the presence of 0.5 mCi/mL+ K; t! \/ `+ m: T) G& g
[3H]thymidine (2 Ci/mmol). Following fixation of cells in
' A+ k* }4 S6 d7 [1 [; z, F; e4% formalin, coverslips were coated with Kodak NTB-2
1 j( S! x+ D% Q2 |! ^( K+ Eemulsion, stored desiccated at 4°C for 3 days, and developed' m7 e8 P3 v& r- `; f2 _% l6 `& A$ D
in D-19 developer and rapid fix (Kodak). Mitotic index, Z! a1 }& j. I
was calculated by counting radiolabeled cells on each coverslip E4 M9 J+ Z" V2 e/ h$ @1 T
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
