Biomedical Engineering Reference
In-Depth Information
are uninfected cells as well as cells incubated with fl uorescent
secondary, but not primary antibodies. A positive control
typically includes cells stained for a normally inaccessible
viral epitope that becomes increasingly exposed over the
course of infection.
8. To achieve rapid binding of the virus to cells, the volume of tissue
culture medium can be reduced by approximately twofold, as
long as the infected cells are completely submerged in the
medium. Alternatively, viruses could be absorbed to cells at 4 °C
in a very low amount of growth medium with gentle agitation to
produce more homogeneous infection kinetics upon subsequent
temperature shift. In addition, since fl ow cytometry is a highly
sensitive quantitative assay, the MOI required to achieve robust
signal intensity is usually much lower than the MOI necessary to
detect virus particles by IF microscopy or PLA. Decreasing the
MOI often translates into increased ability of an assay to detect
small changes in signal intensity. In our hands, fl ow cytometry-
based assays for measuring virus capsid disassembly are superior
to IF-based assays, because the former method eliminates the
inherent bias of cell selection and is intrinsically quantitative and
many more cells can be analyzed. Obviously, however, standard
fl ow cytometry does not provide information regarding subcel-
lular localization of the disassembly event.
9. All subsequent incubations can be carried out at room tem-
perature and the centrifugations performed at 1,000 rpm for
5 min.
10. Do not vortex cells after addition of the primary (or second-
ary) antibody. Instead, gently pipette the cell suspension up
and down several times to avoid splashing of the sample.
Vortexing can be used again during the washing steps.
Acknowledgments
The work in the authors' laboratories on virus entry and infection
is supported by NIH grants CA16038, AI054359, AI062428, and
AI102876.
References
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Engelman A, Xavier RJ et al (2008)
Identifi cation of host proteins required for
HIV infection through a functional genomic
screen. Science 319:921-926
2. Li Q, Brass AL, Ng A, Hu Z, Xavier RJ, Liang
TJ et al (2009) A genome-wide genetic screen
for host factors required for hepatitis C virus
propagation. Proc Natl Acad Sci U S A 106:
16410-16415
3. Karlas A, Machuy N, Shin Y, Pleissner KP, Artarini
A, Heuer D et al (2010) Genome-wide RNAi
screen identifi es human host factors crucial for
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