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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+ [9 V) p7 J) Q
Cell Growth Assays
) O# R0 H2 t0 T& R3 \8 _& x. F+ ZGrowth curves for individual cell lines were established by8 Q( [8 ~' c7 |+ z& e$ q2 l1 ?: J
plating a series of cultures at three different cell concentrations
5 _( M$ k# G* ](i.e., 10^5, 3 X10^4, and 10^4 cells/mL) and calculating
) ^9 l4 O5 _( T# v! B( n4 a& r3 rcell counts at 24-h intervals for 2–3 days. The population
& S6 g+ L4 _, D2 Adoubling time was derived by plotting cell concentration# [) L& n0 q( W3 D
against time. Cell proliferation was also assessed using an
( R: l4 O3 C l+ i7 K6 Wenzyme-linked immunoassay to measure incorporation of
: J2 u2 \; H7 x% P5-bromo-29-deoxyuridine (BrdU) into cellular DNA
5 l& s0 m7 U" [' I t(Boehringer Mannheim). In this assay, cells (4 X10^2) were& v- ^, d: A! Q3 {4 B
cultured on 96-well plates and sequentially incubated with
; K9 W- N+ P f* i+ L. K% _BrdU for 2 h at regular intervals (every 8 or 12 h for 4 days),& `/ I8 t4 v# ^1 X
fixative, and peroxidase-conjugated mouse monoclonal antibody: s* ~9 u# A) e- a6 K
against BrdU. Peroxidase activity was detected using, _+ _# @& O5 Z6 g
tetramethylbenzidine (TMB) as the color substrate. After3 i* N! X0 X0 a0 u' a0 y
color development (' 15 min), absorbance was measured at
( l) Z) w! `1 Z8 S/ I340 nm using a plate reader (Bio-Tech). To establish a& G+ }8 s" | a) g1 s g6 i7 _, P
mitotic index for each cell line, approximately 5 X10^4 cells
/ r1 K# K |9 K9 hwere plated on glass coverslips, allowed to attach, and then
% l- |) p% X8 `incubated for 12–18 h in the presence of 0.5 mCi/mL
9 b! a* i6 Q4 {/ c! k[3H]thymidine (2 Ci/mmol). Following fixation of cells in# } D. H3 r& `: R7 K8 u8 Z8 g
4% formalin, coverslips were coated with Kodak NTB-27 G2 p# Z( X0 w' N% W0 o& H% C
emulsion, stored desiccated at 4°C for 3 days, and developed
9 D$ x/ y2 I% Iin D-19 developer and rapid fix (Kodak). Mitotic index$ e7 W4 S0 p8 ]3 L* ]5 z% \8 B
was calculated by counting radiolabeled cells on each coverslip1 V Y7 s6 c' R: @1 q/ q+ 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
