细胞生长曲线如何测定？

lixiaodong

这是我从一篇文章的Materials and Methods中copy过来的，供参考。
9 a/ I' m" c; `Cell Growth Assays& k$ _8 E- s* `$ j$ @2 T
Growth curves for individual cell lines were established by" T& y: z- n) ^; m5 Z. v5 ~
plating a series of cultures at three different cell concentrations) r" z0 q9 {. I
(i.e., 10^5, 3 X10^4, and 10^4 cells/mL) and calculating
$ f9 i% p8 e" f) I5 e/ l) g" Bcell counts at 24-h intervals for 2–3 days. The population) L( t+ p( p# d) `: G4 R
doubling time was derived by plotting cell concentration7 m+ G7 @" K1 t
against time. Cell proliferation was also assessed using an% a3 X. u# D7 a9 Q# Z5 T* y$ T
enzyme-linked immunoassay to measure incorporation of
* x) U' z* e. E1 m3 N, x1 S5-bromo-29-deoxyuridine (BrdU) into cellular DNA
5 ]% k. e0 h2 Q3 K- A1 @(Boehringer Mannheim). In this assay, cells (4 X10^2) were
) a& X  Z, }4 [* O" c' ?, z. U6 Ccultured on 96-well plates and sequentially incubated with6 r: j2 V7 u( N. m
BrdU for 2 h at regular intervals (every 8 or 12 h for 4 days),+ P" b& x  {) e: v" D. z9 K
fixative, and peroxidase-conjugated mouse monoclonal antibody! S6 T# e- G, B
against BrdU. Peroxidase activity was detected using
+ S  w9 B* ^: ztetramethylbenzidine (TMB) as the color substrate. After) ?: ~, s* N. r/ o
color development (' 15 min), absorbance was measured at  y% o/ d1 m0 q4 Y& q! f
340 nm using a plate reader (Bio-Tech). To establish a) @, t' b0 u4 H8 u( h
mitotic index for each cell line, approximately 5 X10^4 cells) x. G) N  d5 G; y
were plated on glass coverslips, allowed to attach, and then
: _3 _9 N% J. i2 @% w0 oincubated for 12–18 h in the presence of 0.5 mCi/mL' h2 D5 E8 B+ Q
[3H]thymidine (2 Ci/mmol). Following fixation of cells in
3 b2 C  j/ [- o$ k! w0 J4% formalin, coverslips were coated with Kodak NTB-20 Y8 j" q$ q( Z
emulsion, stored desiccated at 4°C for 3 days, and developed$ J& G: t! I4 A* N: ~* C# `; j$ ~
in D-19 developer and rapid fix (Kodak). Mitotic index  V& g+ @' y$ `& I
was calculated by counting radiolabeled cells on each coverslip5 R7 {$ C8 k. ]. F% L2 d" s( F
as a percentage of total cell number.

lixiaodong

回复 3# realinter
: U& |0 m5 V& g$ y/ I8 e5 DThanks.