Biology Reference
In-Depth Information
ponents of such matrices are usually large macromolecules that coat plastic
relatively well; they are also bound with at least moderate affinity by cells. For
these reasons, a concentration range between 1-20
g/mL is usually adequate,
although it is advisable to carry out a range-finding dose-response experiment
before focusing on a narrow range. If a nonmatrix molecule or a complex mixture
is to be tested, a higher concentration should be used. The handling of adhesion
molecules prior to dilution will vary; some molecules, such as fibronectin, are
best thawed quickly at 37
µ
°
C, whereas others, such as laminin, are best thawed
slowly on ice.
4.
Time-course studies have shown substantial coating of proteins onto plastic
within an hour at room temperature and this allows the assay to be performed
quickly. However, if the adhesion molecule binds weakly to plastic, or if it is
more convenient to carry out the experiment the next day, wells can be coated
overnight usually without detrimental effects.
5.
Trypsin, EDTA, or trypsin/EDTA solutions are commonly used to detach adher-
ent cells. The action of these reagents must be terminated prior to using the cells
in spreading assays (e.g., by resuspending the cells in DMEM with 10% [v/v]
fetal calf serum), however, the use of these agents usually has no deleterious
effect on adhesive activity provided the cells are not overtrypsinized. It is impor-
tant to guard against clumping or aggregation of cells, therefore, gentle condi-
tions should be used when centrifuging and resuspending cells. All solutions used
during the preparation of cell suspensions should be warmed to 37
°
C.
6.
Cells are left upright in a polypropylene tube in a humidified 5% humidified CO
2
atmosphere with the lid off to allow them to recover from the process of detach-
ment. Alternatively, the tube can be capped and left on its side in an incubator to
stop the cells from settling and aggregating into a large clump at the bottom of
the tube (they should not be left too long or there may be some nonspecific adhe-
sion to the sides of the tube). The cells should be pipeted gently prior to use to
ensure dispersion. Finally, gassing of the cell suspension with CO
2
can some-
times give enhanced spreading, although this is not always needed. It is impor-
tant that the DMEM/PBS mixture has the opportunity to equilibrate as rapidly as
possible with gaseous CO
2
in order to reestablish the buffer. This process can be
aided by leaving the lid off the microtiter plate in the incubator. It is also our
experience that the adhesion of some cell types is improved by raising the con-
centration of gaseous CO
2
from the usual 5% (v/v) to 7% (v/v). This can be par-
ticularly effective at increasing binding to poorly adhesive substrates. It may also
be advisable to use a particular incubator and/or time of day when the door to the
incubator will not be opened, as this helps prevent alkalinization of the medium.
Ensure that the shelves holding the microtiter plates are horizontal, as uneven
shelves lead to uneven settling of cells.
7.
For attachment assays, the key parameter is the washing protocol as this is the
major determinant of the signal:noise ratio. This is the most critical stage in an
attachment assay, and needs to be optimized for each cell type used. Different
cells respond differently to tapping and washing, and we recommend varying the
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