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1999 Macmillan Magazines Ltd letters to nature
 

Summary: © 1999 Macmillan Magazines Ltd
letters to nature
168 NATURE |VOL 397 |14 JANUARY 1999 |www.nature.com
plated in triplicate into 12-well plates. At various time points cells were stained
with crystal violet (Sigma) and the optical density at 590 nm was determined15
.
Values were normalized to the optical density at day 0 (20 h after plating).
Phoenix producer cells were used to generate retroviral stocks as described15
.
For infection, subcon¯uent passage 1 MEF cultures were incubated at 37 8C
with viral supernatant in the presence of 4 mg ml-1
polybrene (Sigma). After 6 h
the viral supernatant was diluted 1:3 with complete medium and left on the
cells for 42 h. Appropriate dilutions of viral supernatants were used such that
100% of MEFs were infected.
BrdU-incorporation assay and western blotting. MEFs were grown on
coverslips and incubated for 4 h with 10 mM BrdU (Amersham). Cells were
washed in PBS, ®xed for 15 min at -20 8C in 5% acetic acid/95% ethanol and
incubated in PBS. Fixed cells were incubated for 20 min in 2 M HCl/0.5%
Triton-X100, for 30 min in blocking solution (5% fetal calf serum, 5% normal

  

Source: Alon, Uri - Departments of Molecular Cell Biology & Physics of Complex Systems, Weizmann Institute of Science
Batzoglou, Serafim - Department of Computer Science, Stanford University

 

Collections: Biology and Medicine; Biotechnology; Computer Technologies and Information Sciences