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这是我从一篇文章的Materials and Methods中copy过来的，供参考。% \$ p% p- S. ]7 d0 c* U4 n
Cell Growth Assays) s f) a) J3 s. Y3 M. [
Growth curves for individual cell lines were established by
) y) X4 e9 W' B. _" e& Nplating a series of cultures at three different cell concentrations2 O- [- }2 U4 i* h/ O) n
(i.e., 10^5, 3 X10^4, and 10^4 cells/mL) and calculating
# _9 U* L1 L1 K- Icell counts at 24-h intervals for 2–3 days. The population$ K1 |% _) @9 q) `/ h3 ~: @8 t
doubling time was derived by plotting cell concentration8 b2 e: |( M; |, f4 B( o4 r5 j
against time. Cell proliferation was also assessed using an
- m2 ~1 O% Y$ M) W1 t" \7 y8 \6 Menzyme-linked immunoassay to measure incorporation of
- ^2 [+ ^9 K8 ^! _% v5-bromo-29-deoxyuridine (BrdU) into cellular DNA
! K+ k- p% V, k* ?! x2 v(Boehringer Mannheim). In this assay, cells (4 X10^2) were5 u: {" b ~8 F. F- {
cultured on 96-well plates and sequentially incubated with
. }0 ]7 ]6 {1 F$ s1 `6 M/ W4 QBrdU for 2 h at regular intervals (every 8 or 12 h for 4 days),* C, g0 s" E1 k$ d# B
fixative, and peroxidase-conjugated mouse monoclonal antibody
l. h9 p- y, h' y! R4 r; ^against BrdU. Peroxidase activity was detected using
1 g: z1 y4 E" w" a) o+ n O6 ^7 Ytetramethylbenzidine (TMB) as the color substrate. After
: u. ^8 b- u, d+ J" o6 |color development (' 15 min), absorbance was measured at
8 J. k8 _1 H0 V+ J; D: O340 nm using a plate reader (Bio-Tech). To establish a
2 h3 Y6 I. X0 d% Z5 g. Qmitotic index for each cell line, approximately 5 X10^4 cells& R. c4 y& A; s7 x- z! E2 Y$ R
were plated on glass coverslips, allowed to attach, and then3 |! G+ s" ~* |* ~/ }
incubated for 12–18 h in the presence of 0.5 mCi/mL5 M! l1 n' f" W( A. S2 c
[3H]thymidine (2 Ci/mmol). Following fixation of cells in
/ J, M: e9 y5 w6 }! b8 n4% formalin, coverslips were coated with Kodak NTB-23 E6 i9 s) K' Y# k
emulsion, stored desiccated at 4°C for 3 days, and developed
7 s( P" X$ t6 @; {9 z; u6 Nin D-19 developer and rapid fix (Kodak). Mitotic index
: r1 j& I3 B' vwas calculated by counting radiolabeled cells on each coverslip2 X& n/ g2 N0 N3 u: B2 F
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
