This article describes a method for studying the effects of test and control proteins on hippocampal neuron behavior using a silicon matrix with patterned protein zones. The approach allows for the examination of neuronal attachment and growth in response to different protein environments.
Begin with a dish containing a silicon matrix that has multiple small channels connected to inlet and outlet ports.
Inject the test protein with repulsive activity through the inlet to fill the channels.
Incubate to allow protein adherence in the channel area, forming test protein zones, then wash.
Remove the matrix, overlay the coated area with a control protein possessing attractive activity, and incubate.
This facilitates the control protein's adherence to the empty areas between the test protein zones, creating control protein zones with an alternating striped pattern,then wash.
Add laminin to coat the striped area, then wash.
Seed the coated area with a single-cell suspension of hippocampal neurons and incubate.
In the test protein zone, the neurons interact with the proteins, triggering signals that cause the neurons to repel, inhibiting their attachment.
In contrast, neuron-protein interactions in the control protein zone promote neuronal attachment, supporting their growth and axon extension.
Prepare the fluorescently labeled recombinant proteins in PBS. Make 25 microliters for each stripe dish. Let the mixture incubate at room temperature for 30 minutes. Next, for each dish, load a 22-gauge syringe with 25 microliters of diluted protein and inject it through the small hole on the side of the matrix.
Avoid injection of air bubbles into the matrices as they will disturb the binding of your proteins onto the disease.
Now, incubate the dishes for 30 minutes at 37 degrees Celsius in a cell culture incubator. After the incubation, deposit 300 microliters of PBS into the top slit of each matrix. Then, aspirate the PBS from a small hole located on the side of each matrix to remove all the unattached recombinant proteins. Do not fully aspirate the PBS because the proteins will dry out.
Perform this PBS rinse a total of three times. Next, carefully remove the matrix from each dish. Then, immediately add about 100 microliters of control protein over the entire striped area, creating an alternate coating on each dish.
Next, incubate the dishes for 30 minutes at 37 degrees Celsius. Meanwhile, on ice, thaw laminin in PBS at 20 micrograms per milliliter. After 30 minutes, wash the dishes three times with PBS. Then, coat each dish with about 100 microliters of cold laminin and return the dishes to the incubator for another hour.
An hour later, use PBS to wash the dishes three times again. After the third wash, add about 150 microliters of culture medium to each dish. Then, incubate the dishes in a 5% carbon dioxide incubator at 37 degrees Celsius until they are next needed. Count the cells using a hemocytometer and play 10,000 neurons in 150 microliters of culture media on each prepared stripe plate. Carefully deposit the suspension to cover the entire stripe region.
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