268
4 Notes
1. For a detailed description of the light boxes, see refs. 16, 17.
2. It is also possible to use other cell lines. However, be aware
that signal transduction can differ between cell lines and that
the procedure described here might not be directly transferable to other cell lines.
3. When no CASY cell counter is available, a hemocytometer in
combination with a bright field microscope can be used to determine the cell number. If there is also no hemocytometer available,
the amount of cells to be seeded can be vaguely estimated.
One dense 10-cm plate contains approximately 1.5 × 10
7
cells.
4. When rocking the plate try to form a lying eight or perform a
north-south followed by an east-west movement. Avoid circular movement, since this is causing an accumulation of the cells
in the middle of the plate. Once placed in the incubator do not
move the plate for at least a couple of hours to ensure attachment of the cells.
5. To optimize the optoSystem the amount and/or the ratio of
CRY2 and CIBN constructs can be adjusted.
6. Perform same movement as for seeding to prevent local accumulation of transfection mix.
7. The generation of a stable cell line takes time to begin with but
is going to save time later. Furthermore, the overall expression
level of the recombinant proteins can be further refined by cell
sorting.
8. Instead of using a packaging cell line (i.e., Platinum E) the
generation of retroviral vectors can be conducted by transfecting
HEK293T cells with the appropriate helper plasmids.
9. When filtrating viral particles use a filter with low proteinbinding properties. PVDF and nitrocellulose filter will bind
viral particles and strongly reduce transduction efficiency.
10. Wrap plates carefully with parafilm to prevent accidental splashing of viral particles and to delay equilibration of CO 2 with
surrounding.
11. In case trypsin-sensitive cells are used it may be advisable to
centrifuge the cells and discard the supernatant to remove all
trypsin; alternatively, an enzyme-free detachment solution can
be used.
12. It is possible (and a good idea) to expand the stable cell line
generated at this point or after a first experiment, ensuring
proper functionality of the optoSystem. This allows skipping
future work under BSL2 conditions and also provides a backup
of cells.
Wignand W.D. Mühlhäuser et al.
4 Notes
1. For a detailed description of the light boxes, see refs. 16, 17.
2. It is also possible to use other cell lines. However, be aware
that signal transduction can differ between cell lines and that
the procedure described here might not be directly transferable to other cell lines.
3. When no CASY cell counter is available, a hemocytometer in
combination with a bright field microscope can be used to determine the cell number. If there is also no hemocytometer available,
the amount of cells to be seeded can be vaguely estimated.
One dense 10-cm plate contains approximately 1.5 × 10
7
cells.
4. When rocking the plate try to form a lying eight or perform a
north-south followed by an east-west movement. Avoid circular movement, since this is causing an accumulation of the cells
in the middle of the plate. Once placed in the incubator do not
move the plate for at least a couple of hours to ensure attachment of the cells.
5. To optimize the optoSystem the amount and/or the ratio of
CRY2 and CIBN constructs can be adjusted.
6. Perform same movement as for seeding to prevent local accumulation of transfection mix.
7. The generation of a stable cell line takes time to begin with but
is going to save time later. Furthermore, the overall expression
level of the recombinant proteins can be further refined by cell
sorting.
8. Instead of using a packaging cell line (i.e., Platinum E) the
generation of retroviral vectors can be conducted by transfecting
HEK293T cells with the appropriate helper plasmids.
9. When filtrating viral particles use a filter with low proteinbinding properties. PVDF and nitrocellulose filter will bind
viral particles and strongly reduce transduction efficiency.
10. Wrap plates carefully with parafilm to prevent accidental splashing of viral particles and to delay equilibration of CO 2 with
surrounding.
11. In case trypsin-sensitive cells are used it may be advisable to
centrifuge the cells and discard the supernatant to remove all
trypsin; alternatively, an enzyme-free detachment solution can
be used.
12. It is possible (and a good idea) to expand the stable cell line
generated at this point or after a first experiment, ensuring
proper functionality of the optoSystem. This allows skipping
future work under BSL2 conditions and also provides a backup
of cells.
Wignand W.D. Mühlhäuser et al.