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 ?6 n% r, f. w. y# T
Cell Growth Assays
. j% i! m5 P) G$ j( l; `1 @Growth curves for individual cell lines were established by5 G# H# s, l6 P0 Q  s* \# }! z
plating a series of cultures at three different cell concentrations$ l% Q- M6 z3 C; Q- i* }: {
(i.e., 10^5, 3 X10^4, and 10^4 cells/mL) and calculating) ~/ w& R7 M; g9 Z/ o
cell counts at 24-h intervals for 2–3 days. The population2 @) ^% {: s( @2 j8 c4 f
doubling time was derived by plotting cell concentration
( ^5 F. L2 Z  r  i! `2 |5 uagainst time. Cell proliferation was also assessed using an
' E. [4 b$ x1 yenzyme-linked immunoassay to measure incorporation of
, n+ D4 _5 b( N5 j5-bromo-29-deoxyuridine (BrdU) into cellular DNA
) m' r4 I, F2 Y' y4 [8 ~4 Z8 E6 ?# |(Boehringer Mannheim). In this assay, cells (4 X10^2) were) o, Y! F# Y; }. Y' o" ]0 X& X
cultured on 96-well plates and sequentially incubated with& x( E4 F9 u! `5 L" j& G
BrdU for 2 h at regular intervals (every 8 or 12 h for 4 days),* c; {  J* Z! {7 O# `6 f$ j3 ~
fixative, and peroxidase-conjugated mouse monoclonal antibody, T, y7 W5 ^& g# u0 S9 G
against BrdU. Peroxidase activity was detected using
6 i# n1 f1 F& p" G2 V) G0 A1 K% {- G6 Qtetramethylbenzidine (TMB) as the color substrate. After" R5 A1 x2 p; I" C* A
color development (' 15 min), absorbance was measured at
/ G1 w: F! Q. X$ Q. t) {$ V340 nm using a plate reader (Bio-Tech). To establish a
- G7 ?) F. L+ h9 Fmitotic index for each cell line, approximately 5 X10^4 cells0 F. }% n8 s$ x  r' ^, C
were plated on glass coverslips, allowed to attach, and then, |1 T4 P" A; M. X
incubated for 12–18 h in the presence of 0.5 mCi/mL
  G; O. y9 D" i% z- X: X  E[3H]thymidine (2 Ci/mmol). Following fixation of cells in
+ V7 U1 K6 U( ~0 x2 I8 X* L4% formalin, coverslips were coated with Kodak NTB-2
3 O& h3 P3 [% t% p: z9 oemulsion, stored desiccated at 4°C for 3 days, and developed8 s1 U% F  b) i2 b# \
in D-19 developer and rapid fix (Kodak). Mitotic index
8 _8 h* h, K8 S/ j/ y) b* g# ~was calculated by counting radiolabeled cells on each coverslip
+ |) m6 n/ T# k' F9 f4 ~as a percentage of total cell number.

lixiaodong

回复 3# realinter " B0 L: q4 }; V  z: c5 `' M
Thanks.