这是我从一篇文章的Materials and Methods中copy过来的，供参考。 + P' |/ D1 i$ z3 i, DCell Growth Assays' c9 `6 I( G5 Y- O y$ ^7 w0 `# [
Growth curves for individual cell lines were established by 8 W( E0 l) f {$ n3 N8 Wplating a series of cultures at three different cell concentrations) M( E1 Y5 |7 s9 @/ q( \
(i.e., 10^5, 3 X10^4, and 10^4 cells/mL) and calculating ' S) w7 X8 s9 h4 {6 e2 [$ Ucell counts at 24-h intervals for 2–3 days. The population - E1 D/ l6 b. C. ndoubling time was derived by plotting cell concentration% {% R+ \/ U' Z# s1 [4 l* Y
against time. Cell proliferation was also assessed using an 6 @! e" n6 v7 _3 `& o% x& o& @enzyme-linked immunoassay to measure incorporation of % X( X+ J0 w, z* c3 O' ]5-bromo-29-deoxyuridine (BrdU) into cellular DNA: ~/ B! g A0 f- G
(Boehringer Mannheim). In this assay, cells (4 X10^2) were # Q, b! b2 ~0 S, s2 c! [cultured on 96-well plates and sequentially incubated with$ [- _0 H7 g1 t8 m( X" `
BrdU for 2 h at regular intervals (every 8 or 12 h for 4 days),! Y$ U6 {# x5 s2 [
fixative, and peroxidase-conjugated mouse monoclonal antibody 2 Z2 c' z& Y4 M5 aagainst BrdU. Peroxidase activity was detected using 8 x' n; ^& W5 N8 {6 Ttetramethylbenzidine (TMB) as the color substrate. After , a7 b6 F# Q8 I" l' }/ Xcolor development (' 15 min), absorbance was measured at2 w. D: c0 J, j, l( I
340 nm using a plate reader (Bio-Tech). To establish a 6 @; f9 ]" o2 G8 ^mitotic index for each cell line, approximately 5 X10^4 cells , c2 n; i( \% R" c4 f- O* ~8 Swere plated on glass coverslips, allowed to attach, and then$ O) ^, R) W! h$ [, m9 l0 _& {- @
incubated for 12–18 h in the presence of 0.5 mCi/mL * y! \. U: f& U7 e& A. \1 k[3H]thymidine (2 Ci/mmol). Following fixation of cells in - [# B g1 u0 H" l% _1 M4 z% A4% formalin, coverslips were coated with Kodak NTB-2 3 l) q9 P; E9 wemulsion, stored desiccated at 4°C for 3 days, and developed; W3 E7 a+ K% R6 S
in D-19 developer and rapid fix (Kodak). Mitotic index 4 v+ }: `, J( P) Nwas calculated by counting radiolabeled cells on each coverslip . ? k0 a \& n- a$ Zas a percentage of total cell number.作者: realinter 时间: 2010-8-2 17:08
