Cell Adhesion Assays - Methods in Molecular Biology: Extracellular Matrix Protocols - page 268

standing of the signaling mechanisms that control cell morphology has
improved, spreading assays can give indirect indications of the intracellular
events that are triggered by certain substrates. A further advantage of spread-
ing assays is that they are more sensitive when used to measure inhibitory
activity because the read-out from the assay is more reliant than attachment
assays on multiple adhesive interactions and partial disruption by an inhibitor
is sufficient to see blockade. Finally, because spreading assays do not need
replicate wells, they are more economical in their use of substrates. As
described below, both types of adhesion assay are relatively quick to carry out.
The actual assays can be performed in half a day, however the quantitation of
spreading assays can take a similar length of time.
Sometimes there is no alternative to an attachment assay, because some cells
are unable to spread at all, whereas other cells can only spread on specific
substrates. Although attachment assays still require multiple cell-substrate con-
tacts to allow the cell to withstand the washing steps in an attachment assay,
fewer contacts are needed than for a cell to spread.
Both the spreading and attachment assays are expressed as percent adhesion
and it can be expected that the level of adhesion obtained will vary depending
upon the cell type and adhesive substrate under study. In spreading assays, a
level of 80% is common, and often higher levels can be obtained. Most impor-
tantly, the background level of spreading on BSA-coated plastic should be as
low as possible. Frequently, this is actually zero, but certainly this should not
rise above a few percent. The level of attachment observed by the dye-staining
method is usually not as high as for spreading, but 60-70% should be attainable.
A low figure for the BSA background is also important for attachment assays,
but generally it is difficult to reduce this level below 5% without adversely
affecting the experimental signal ( see Note 1 ).

2. Materials
2.1. Common to Both Assays
1. 10 mg/mL heat-denatured BSA in divalent cation-free Dulbecco's PBS. This is a

better blocking agent than native BSA solutions. After dissolving the BSA, filter
through a 0.22- µ m filter to remove undissolved protein, and incubate in a water

bath at 85 C for 10-12 min. The solution should be slightly hazy; it should not be

clear, as this will contain insufficiently aggregated BSA, nor should it be white,
because here the aggregates will be too large. After cooling, it is ready for use.
Occasionally, some cell types find heat-denatured BSA toxic and therefore care
should be taken to wash the wells of the microtiter plate after blocking.

°

2. 96-well tissue-culture-treated microtiter plate (Costar, Cambridge, MA) ( see

Note 2 ).

3. Dulbecco's PBS (Gibco-BRL, Gaithersburg, MD).