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这是我从一篇文章的Materials and Methods中copy过来的，供参考。% `; ~+ w! j8 a/ s, t
Cell Growth Assays* x. [ i4 F# i* q V
Growth curves for individual cell lines were established by
1 B ]3 L2 L* p+ |7 F' L+ p. Lplating a series of cultures at three different cell concentrations. z2 H* A; R3 u5 p* R, C
(i.e., 10^5, 3 X10^4, and 10^4 cells/mL) and calculating
/ t$ o r9 I$ \) O) I' o. p4 lcell counts at 24-h intervals for 2–3 days. The population
: R6 n$ m8 h- Y. Z- S+ Sdoubling time was derived by plotting cell concentration; h5 H1 }" I2 P3 Q7 L A# |
against time. Cell proliferation was also assessed using an
" U' q( G0 r, \8 @8 benzyme-linked immunoassay to measure incorporation of* ~' h( }1 q$ I3 t7 T
5-bromo-29-deoxyuridine (BrdU) into cellular DNA
. n) Z" s" u2 |/ k# z9 }(Boehringer Mannheim). In this assay, cells (4 X10^2) were: q; P! H3 c* i7 U& B
cultured on 96-well plates and sequentially incubated with
! x0 H) n/ h8 [" e9 s: HBrdU for 2 h at regular intervals (every 8 or 12 h for 4 days),
/ t+ G1 ~' x7 `# Nfixative, and peroxidase-conjugated mouse monoclonal antibody! w6 O% T/ \7 S$ S9 i: c2 i' Y$ y
against BrdU. Peroxidase activity was detected using- O- Z* R% {1 n; H; S
tetramethylbenzidine (TMB) as the color substrate. After4 n7 F/ `; j( Q
color development (' 15 min), absorbance was measured at
- |8 u' r/ L- ~$ ^! ]2 S340 nm using a plate reader (Bio-Tech). To establish a( X5 L8 [1 a0 G: a9 ^- L# i! u- _
mitotic index for each cell line, approximately 5 X10^4 cells- I0 M6 _8 H0 z: R4 G
were plated on glass coverslips, allowed to attach, and then/ P$ l9 H8 t3 R
incubated for 12–18 h in the presence of 0.5 mCi/mL
2 o3 Z& ^3 ^4 ]$ G# U[3H]thymidine (2 Ci/mmol). Following fixation of cells in
* j! g0 u) [/ z$ E4% formalin, coverslips were coated with Kodak NTB-2
- G5 @2 R* ~& d# kemulsion, stored desiccated at 4°C for 3 days, and developed7 L4 v0 p8 Y& T! Q2 n
in D-19 developer and rapid fix (Kodak). Mitotic index9 O( G% a* a" [( Y( T
was calculated by counting radiolabeled cells on each coverslip
; V9 I; `5 U6 [0 O' qas 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
