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这是我从一篇文章的Materials and Methods中copy过来的，供参考。
( @1 o- s0 @9 GCell Growth Assays8 M+ Z1 P# c m( k6 I3 M
Growth curves for individual cell lines were established by) ]4 Y* ]. Z& x( N/ s" G* U5 S5 n
plating a series of cultures at three different cell concentrations
% _+ l2 ?4 W$ J2 ?4 p, {, m(i.e., 10^5, 3 X10^4, and 10^4 cells/mL) and calculating7 ^+ h2 l0 f. y3 B
cell counts at 24-h intervals for 2–3 days. The population
* ]/ G/ D+ C2 q! E" Ddoubling time was derived by plotting cell concentration
: Y7 Z" p' P% m6 Y! o( @. lagainst time. Cell proliferation was also assessed using an M6 ]+ a0 v& S+ g: `. I4 u
enzyme-linked immunoassay to measure incorporation of
6 u. M0 p# L) z5-bromo-29-deoxyuridine (BrdU) into cellular DNA
( O5 s7 b: F2 _8 L( n/ a- I(Boehringer Mannheim). In this assay, cells (4 X10^2) were P1 i9 o6 J& `6 i( P6 d
cultured on 96-well plates and sequentially incubated with
; M" ]- j3 r' e+ n+ v6 {; }BrdU for 2 h at regular intervals (every 8 or 12 h for 4 days), ?7 A8 i8 O' J! Z/ b0 S
fixative, and peroxidase-conjugated mouse monoclonal antibody+ a! ?. | u# w! q
against BrdU. Peroxidase activity was detected using
+ D% H8 Z2 |' m" Ktetramethylbenzidine (TMB) as the color substrate. After' m# X: Y) e9 d) n! b* Q4 M, N
color development (' 15 min), absorbance was measured at) |# t4 B+ u* t* b
340 nm using a plate reader (Bio-Tech). To establish a. N: ]+ T U+ I# x3 D; z
mitotic index for each cell line, approximately 5 X10^4 cells7 c' m M" r) u$ R. ?9 x, W
were plated on glass coverslips, allowed to attach, and then9 U# u1 }' r! o
incubated for 12–18 h in the presence of 0.5 mCi/mL3 a( \# Z" W; q7 u: P/ U& s& H( r
[3H]thymidine (2 Ci/mmol). Following fixation of cells in9 T5 O1 u- @9 n* p) Q
4% formalin, coverslips were coated with Kodak NTB-24 ]" o# S* h! w0 X* v4 E& w5 c1 w+ r, T
emulsion, stored desiccated at 4°C for 3 days, and developed
2 q& |1 T5 `9 x5 Xin D-19 developer and rapid fix (Kodak). Mitotic index
5 ?/ r0 h3 F& N( u. `3 E* Mwas calculated by counting radiolabeled cells on each coverslip
/ U o, J' V! ~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
