A major application of cell adhesion assays is in investigating whether a certain cell type can adhere to a specific adhesive substrate, and, if so, which receptors are involved. Particularly if the substrate is a matrix component e. Procedures are described here for assessing which integrins are involved in this process. Although peptides and other inhibitors have, in general, less specificity than monoclonal antibodies, they can be useful for determining what amino acid sequence in the ligand is recognized by the integrin.
See above list of suppliers for antibodies to other species. For cell attachment assay : Mix an aliquot of the cell suspension with an equal volume of DPBS containing the inhibitor at twice the final concentration. However, the concentration of mAb to achieve a maximal level of inhibition should be tested using a range of mAb concentrations. Many commercially available mAbs contain sodium azide as a preservative.
This is toxic to all cells and will inhibit attachment and spreading. Therefore, it is essential that the sodium azide be removed by dialysis into an appropriate buffer, such as DPBS. Peptides can be used in place of mAbs to analyze the contribution of integrins to cell adhesion. However, these are generally less useful than mAbs because only for a small number of integrins have specific inhibitor peptides been described.
RGD peptides e. Concentrations of peptides required to inhibit cell adhesion are generally higher than for mAbs: typically 0. Higher concentrations are normally required for inhibiting cell attachment than for inhibiting cell spreading.
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Incubate, fix, and analyze for spreading assay see Humphries, , Basic Protocol 1 , steps 8 to 13 or for attachment assay see Humphries, , Basic Protocol 2 , steps 8 to It is essential that the effect of each mAb on cell attachment or spreading be compared with appropriate controls. The best controls to use are mAbs that are noninhibitory against the integrins under test—e. Control peptides e. Integrins can be purified by detergent extraction from tissue or from cell lines; alternatively, they can be made recombinantly e. Leave a set of wells empty for measuring binding of the ligand to BSA.
The authors normally perform the assay using four replicates for each sample. A small amount of blocking solution is added to the wells before aspirating the integrin solution because the authors have found that this renders the wells hydrophilic and prevents them from drying out when they are aspirated.
Drying out of the wells destroys the activity of some of the integrin. Remove the buffer by aspirating or flicking it out. Repeat two times.
Remove residual liquid by inverting the plate and striking it hard several times onto absorbent paper towels. The appropriate concentration must be determined by pilot experiments.
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A concentration of 0. Other reagents e. By using a range of inhibitor concentrations at a constant ligand concentration , IC 50 values can be determined see e. A cell culture incubator is suitable for this incubation.
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Flick out or aspirate solutions from the wells to remove unbound ligand. Remove the residual buffer see step 7. During this time prepare the ABTS reagent. Aspirate solutions from the wells to remove unbound ExtrAvidin peroxidase reagent.go to link
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Calculate the mean and standard deviation of the absorbance readings for ligand binding to integrin, and for ligand binding to BSA alone, using the following equations. The procedure can be adapted to purify other integrins from different tissue sources or from pellets of cultured cells. CAUTION : Human placenta should be treated as potentially biohazardous; take suitable precautions, such as wearing latex gloves, eye protection, and a laboratory coat. Commercially available laboratory homogenizers may be used, but a robust domestic blender is adequate for this purpose.
Thaw the homogenate in a cold room preferable or at room temperature overnight.
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Centrifuge again under the same conditions and discard the supernatant as described. Most of the soluble proteins have been removed at this stage. The extraction buffer contains protease inhibitors and BSA to minimize proteolytic degradation of the integrins. The extract obtained will be red in color because not all of the hemoglobin has been removed in step 3. Preclear and filter the supernatant from step 5 by passing it through the column and collecting the flowthrough.
Several columns can be used simultaneously to speed up this step. This step removes proteins that bind to rat IgG. The authors normally discard the rat IgG—Sepharose at this stage. Mix the fractions with this buffer as they elute from the column to ensure prompt neutralization. Neutralize the column immediately with 20 ml of 0. Because of the low pH used to elute the integrin, the mAb 13—Sepharose gradually deteriorates in its capacity for integrin purification. However, in our experience the column can be reused about ten times before replacement is necessary.
Destain the gel and check for elution of integrin. A typical elution profile is shown in Figure 1. Pack the suspension into a 0. Collect 0.
Neutralize the column with 5 ml of 0. Both mAbs are available Millipore Sigma. Measure the absorbance of an aliquot of the dialyzed mAb at nm. Determine the absorbance at nm for an aliquot of the recovered supernatant and compare to the starting value according to the following equation, where D is the dilution factor total volume divided by volume of starting solution. The buffer for washing may be added in several aliquots if the capacity of the funnel is low. Biotin is covalently conjugated to amino groups on the ligand to permit quantification of bound ligand.
Ligand of interest Coupling buffer: 0. About 0. This removes any large aggregates or precipitate from the solution. Measure the concentration of biotinylated protein in the supernatant using, e. ABTS buffer : 0. Add 0. Mix thoroughly. BSA is then added. BSA is then added and dissolved by vigorous stirring. The solution is then centrifuged and filtered as above.