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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