Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

The overall goal of this procedure is to test the ability of protein ecto domains to mediate homophilic adhesion to achieve this plasmids encoding the fusion of an ecto domain fragment to the FC region of human IgG are transfected into HEC 2 93 cells. The secreted Odin FC fusion proteins are captured on magnetic beads conjugated to protein G, and following a wash, the coated beads are allowed to interact in solution. The coated beads are then imaged to assess the extent of aggregation and finally, quantified Using simple image analysis, the resulting data reveal whether the protein understudy is capable of mediating homophilic adhesion.

The main advantages of this technique over existing methods such as cell aggregation assays, is that it is quick, simple, and directly tests the adhesive activity of your chosen protein. Demonstrating this technique will be Dr.Michelle Eman, a senior scientist from the laboratory. Begin this procedure by preparing the cultured cells for NCA here ECFC, two to three days prior to transfection.

Use 0.05%trips in EDTA to detach the HEC 2 93 cells from culture dishes. Prepare two 100 millimeter dishes for each condition. Split the cells one to five, and then culture them in.

Supplemented growth medium. Incubate the cells at 37 degrees Celsius with 5%carbon dioxide until they are 60 to 80%confluent. On the day of the transfection, use a reagent such as lipo to introduce a plasmid encoding FC fusion.

After the transfection, return the cells to the incubator for 24 hours the day after the transfection Prewarm, DMEM with pent strep and without fetal bovine serum to 37 degrees Celsius. Rinse each dish two times with 10 milliliters of the warmed medium, and then return the dishes to the 37 degree Celsius incubator for one hour after the incubation, rinse the culture dishes one more time with 10 milliliters of DMEM without fetal bovine serum for a total of three washes, then incubate the transected cells at 37 degrees Celsius for another 48 hours before collecting the medium. Four days after transfection, use a serological pipette to collect the medium from each pair of culture dishes and transfer it to a 50 milliliter conical tube.

Then centrifuge the medium at 500 Gs per five minutes to pellet the cellular debris after the spin, pour the medium from each 50 milliliter conical tube into a 30 milliliter syringe and filter it through a 0.45 micron syringe. Filter into a centrifugal concentrator. Spin the centrifugal filters until the volume of concentrated culture medium has been reduced to approximately 500 microliters.

This takes approximately 15 minutes. Repeat this process until all of the culture medium has been added and concentrated. Next, in a 1.5 milliliter micro centrifuge tube for each sample at 1.5 microliters of protein G magnetic beads to one milliliter of ice cold binding buffer.

The most difficult aspect of this procedure is the bead aggregation. To ensure success, be sure to follow the procedure exactly. Watch the beads quickly, but not too harshly.

They should never be allowed to dry on the side of the tube or to remain on the magnet too long. Place the tube on a magnet and using a pipette, aspirate the buffer. Then immediately add the concentrated culture medium to the protein G magnetic beads.

Rotate the tubes at four degrees Celsius for two hours. Place the tubes on a magnet and remove the medium quickly. Wash the beads twice with one milliliter of ice cold binding buffer, and then resuspend the beads in 300 microliters of binding buffer.

Split the resuspended beads into two tubes with 150 microliters into each tube. Then add 1.5 microliters of calcium chloride or EDTA for the calcium and no calcium conditions respectively. Transfer 100 microliters from each condition to a depression.

Well slide using a transmitted light microscope. Collect images from five fields of view for each experiment at each desired time point. Using image J or comparable image analysis software.

Open one of the five image data sets in image J in the dropdown menu. At the top of the screen, select file. Then select import, then image sequence.

Find the folder containing the image files, then open them as an image stack. Next in the dropdown menu bar, click on image, then select properties. To open the properties window there.

Change the image units to pixels and set the pixel width and pixel height to 1.0. Click on image again, select adjust then threshold to open the threshold window and use the slider to adjust the image threshold. Include pixels that contribute to beads and exclude and small particles.

Apply the threshold to all of the images in the image stack. Next in the dropdown menu bar at the top of the screen. Click on analyze.

Then select set measurements to open the measurement dialogue box using the check boxes select area and stack position at the top of the screen. Click on analyze, then select analyze particles. This will generate a list of identified particles including their size and the image in which they were identified.

Repeat this process for each experiment and each experimental condition to determine the calcium dependent adhesive ability of zebrafish and cadherin. HEC 2 9 3 cells were transfected with the NCAT herrin OD domain fused two fc and following calcium dependent bead aggregation cells were imaged and analyzed as described in this video as can be seen in these images. In the absence of calcium, the beads exhibit little or no tendency to aggregate, and there is no increase in aggregate size with time in the presence of calcium.

However, beats coated with NCAT here and ECFC show robust aggregation with aggregate size increasing over time as a semi-quantitative measure of adhesion. The transmitted light images from three experiments were analyzed to determine the mean size of the aggregates. As can be seen in this plot, the aggregation of ncoer ECFC coated beads in the presence of calcium and in the absence of calcium was determined at 15 minute intervals.

Over the course of an hour, these data reveal that the ecto domain of zebrafish n cadherin mediates calcium dependent homophilic adhesion. After watching this video, you should have a good understanding of how to test whether adhesion is an intrinsic biochemical property of your protein of interest.