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这是我从一篇文章的Materials and Methods中copy过来的，供参考。. S4 ~# A7 F& e. [' u( } L
Cell Growth Assays' w- w' K, X u+ V7 G
Growth curves for individual cell lines were established by3 |5 ?9 i4 J' t2 g3 ]5 F
plating a series of cultures at three different cell concentrations
' ~- \2 c" G$ y% z(i.e., 10^5, 3 X10^4, and 10^4 cells/mL) and calculating7 I+ S# l) N' {! ^2 K3 v
cell counts at 24-h intervals for 2–3 days. The population
% k2 o; `6 i: H5 R" R0 fdoubling time was derived by plotting cell concentration. r8 f( Y" Y# I
against time. Cell proliferation was also assessed using an
1 a* T$ ]( C! M8 ienzyme-linked immunoassay to measure incorporation of; h9 s" I7 F) {+ F
5-bromo-29-deoxyuridine (BrdU) into cellular DNA) Y. X9 f8 M$ @0 U% x! R
(Boehringer Mannheim). In this assay, cells (4 X10^2) were% r9 z6 G$ Y4 Q$ b0 B$ r
cultured on 96-well plates and sequentially incubated with
+ h3 r- f! e+ [- y7 C7 u4 W8 WBrdU for 2 h at regular intervals (every 8 or 12 h for 4 days),, ]- m* I% Z' I
fixative, and peroxidase-conjugated mouse monoclonal antibody
8 n% p. q* W5 Eagainst BrdU. Peroxidase activity was detected using
) {# B' z. q+ y7 T! Gtetramethylbenzidine (TMB) as the color substrate. After
0 j/ Z* @" V3 [6 N$ Q2 q! t9 k: Acolor development (' 15 min), absorbance was measured at l( q. Z9 F4 H& ~ z/ F% ~
340 nm using a plate reader (Bio-Tech). To establish a; ^8 }/ s0 {5 p" ?8 ~/ W6 _
mitotic index for each cell line, approximately 5 X10^4 cells& O) l2 S! y' Q: a9 }
were plated on glass coverslips, allowed to attach, and then3 I6 ` w" A* J' g2 g# R- R0 F
incubated for 12–18 h in the presence of 0.5 mCi/mL
5 v6 Y0 j/ i( W( b% U[3H]thymidine (2 Ci/mmol). Following fixation of cells in# ?& g% V+ b) M; |2 K$ n- A* C
4% formalin, coverslips were coated with Kodak NTB-25 A$ Q; E. X1 H8 ~: h* a: ?' t
emulsion, stored desiccated at 4°C for 3 days, and developed+ @$ r% U0 m9 A" G) B6 s/ _
in D-19 developer and rapid fix (Kodak). Mitotic index; `' a$ I# i% `' @$ a) H
was calculated by counting radiolabeled cells on each coverslip" G$ W/ H# E1 u6 s# J% H9 Y L3 T
as a percentage of total cell number. |
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发表于 2010-9-13 09:33
发表于 2010-8-2 17:08
