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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 L5 f4 d/ e4 q8 r; u
Cell Growth Assays
- p* j! R% K8 T+ DGrowth curves for individual cell lines were established by* W: T/ F2 I/ E( n4 W
plating a series of cultures at three different cell concentrations
* H% W5 T" }; o* r( s0 C(i.e., 10^5, 3 X10^4, and 10^4 cells/mL) and calculating3 e% m4 }# n3 S+ p. n0 U& ]
cell counts at 24-h intervals for 2–3 days. The population V3 T+ o& x; e* ?
doubling time was derived by plotting cell concentration
, v6 J8 z& u- J9 ^) `* |against time. Cell proliferation was also assessed using an
7 ~$ Q! \9 A* ~- n1 x+ k; Henzyme-linked immunoassay to measure incorporation of
7 C8 R& l- M4 N7 _" O5-bromo-29-deoxyuridine (BrdU) into cellular DNA
d9 \" O% N( Q- `+ a/ T3 x(Boehringer Mannheim). In this assay, cells (4 X10^2) were
1 x0 j7 c T+ ucultured on 96-well plates and sequentially incubated with
4 F" y$ E6 Y+ P) x( iBrdU for 2 h at regular intervals (every 8 or 12 h for 4 days),, Q. {$ h9 j. I: |8 B
fixative, and peroxidase-conjugated mouse monoclonal antibody
3 r* R, g5 ?& S, j4 L- o5 X) G* Magainst BrdU. Peroxidase activity was detected using
8 m$ }) h+ a( [& q s5 Itetramethylbenzidine (TMB) as the color substrate. After
( T4 \" w* \4 T2 Ycolor development (' 15 min), absorbance was measured at
6 |4 c' b4 d+ C340 nm using a plate reader (Bio-Tech). To establish a4 {2 B8 Q0 s2 o5 x. C) C) d" Z
mitotic index for each cell line, approximately 5 X10^4 cells3 F$ P! ~- s1 }" I
were plated on glass coverslips, allowed to attach, and then1 }, `" w- l7 i+ B/ I' j
incubated for 12–18 h in the presence of 0.5 mCi/mL
& V: w+ |5 @! P! y; `[3H]thymidine (2 Ci/mmol). Following fixation of cells in
) Q% N6 v5 {6 p$ k+ X$ m8 n4 H4 Q4% formalin, coverslips were coated with Kodak NTB-2' a2 W$ p7 T. j3 u" n5 E8 |# F
emulsion, stored desiccated at 4°C for 3 days, and developed
; k0 ~* c' D" c3 ~6 X, j P! s% U: }in D-19 developer and rapid fix (Kodak). Mitotic index7 i' t2 Q9 d/ l" F- g
was calculated by counting radiolabeled cells on each coverslip e& i/ C- l0 e; }4 d3 P1 V3 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
