Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons

Axon guidance molecules regulate neuronal migration and targeted growth-cone navigation. We present a powerful method, the stripe assay, to assess the ability of guidance molecules to attract or repulse neurons. In this protocol, we demonstrate the stripe assay by showing FLRT2's ability to repel cultured hippocampal neurons.

The overall goal of the stripe assay is to asses the ability of specific proteins, such as guidance molecules, to attract or repel neurons. This method can help answer key questions in the field of axon guidance, such as, how neurons can migrate or extend their axons in response to the external cellular environment. The main advantage of this technique is that a large number of neurons can be visualized in a single experiment and many growth cones can be analyzed simultaneously.

In preparation, boil four to eight silicone matrices in water for five minutes. Allow them to completely air dry under a laminar flow hood with their striped sides up. This takes about an hour.

Continue working under the hood. Use compressed air or sticky tape to remove any dust on the striped side of the matrices. Then carefully place each matrix into a separate six centimeter plastic dish, striped side down.

With one finger, press each matrix firmly. Avoid trapping air bubbles between the matrix and the dish. If the matrix fails to attach, repeat the process.

Once attached, mark the location of the stripes on the bottom side of the culture dish. Prepare the fluorescently labeled recombinant proteins in PBS. Make 25 microliters for each striped 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 the injection of air bubbles into the matrices as they will disturb the binding of your proteins onto their dishes.

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 controlled 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 and 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. After euthanizing a donor E15.5 mouse, cut the skin and skull sagittally at the mid-line of the head with surgical scissors and scoop the brain using a micro spatula.

Carefully transfer the brain to a 60 milliliter petri dish containing three milliliters of HBSS. In the dish, use a scalpel to cut out the hemispheres including the cortex, hippocampus and striatum. Then dissect out the hippocampal regions by carefully removing the meninges.

Transfer the dissected hippocampal tissues to a 15 milliliter centrifuge tube containing two milliliters of HBSS solution on ice. Six hippocampi from three embryos are sufficient for culturing neurons on eight dishes. With all of the hippocampus tissues collected, aspirate out the HBSS and replace it with two milliliters of trypsin-EDTA.

Then incubate the tube for 15 minutes in a 37 degree Celsius water bath. Next, neutralize the trypsin activity by adding 500 microliters of fetal bovine serum. Then centrifuge the mixture at 100 Gs for five minutes.

Remove the supernatant and wash the pellet with two milliliters of culture medium. Then centrifuge the mixture again and replace the medium with more medium. Now dissociate the tissue with gentle trituration using a one milliliter pipe head with a cut tip.

Flow the tissues up and down through the pipe head tip about 10 times. Then use an uncut one milliliter tip and repeat the trituration to obtain a single cell suspension. Now isolate the single cells by pressing the suspension through an 80 to 100 micron mesh cell strainer.

Then centrifuge the filtered suspension at 150 Gs for five minutes, aspirate out the supernatant and re-suspend the pellet in two milliliters of culture media. Count the cells using a hemocytometer and plate 10, 000 neurons in 150 microliters of culture media on each prepared stripe plate. Carefully deposit the suspension to cover the entire striped region.

Dissociated hippocampal neurons from E15.5 mice were plated and cultured for 24 hours on stripes of flourescently labeled control protein FC, or the neurons were plated on stripes of FLRT2 FC alternating with non-labeled control FC.In both cases, the neurons were aggregated and extended their axons as bundles. On the FC control the neurons were distributed evenly and extended their axons in random directions. In contrast, when cultured with FLRT2 FC stripes, the axons avoided growing on the FLRT2 FC regions.

Thus the extending axons grew mainly on the FC stripes. Higher magnification shows that the axons grew along the border with FLRT2 FC stripes, but did not extend into the FLRT2 territory. After watching this video you should have a good understanding of how to make striped carpet of the recommended proteins and asses the attractive or paras activity using the dissociated hippocampal neurons.

Once mastered, this technique can be done in five hours if it is performed properly.