Purification of the Dendritic Filopodia-rich Fraction

In this protocol, we introduce a method for purifying the dendritic filopodia-rich fraction from the phagocytic cup-like protrusion structure on cultured hippocampal neurons by taking advantage of the specific and strong affinity between a dendritic filopodial adhesion molecule, TLCN, and an extracellular matrix molecule, vitronectin.

Transcript

Compared to PSD fraction, it can be possible to identify the synaptic protein acting on the immature synapse around the dendritic filopodia-rich fraction. We used live neuron to induce phagocytic cap formation. The main advantage of this technique is that active protein in artificial genesis could be identified from the dendritic filopodia rich fraction.

To begin, prepare 200x Vitamin Mix by dissolving 100 mg of Vitamin B5, 100 mg of Choline Chloride, 100 mg of Folic Acid, 180 mg of I-inositol, 100 mg of Vitamin B3, 100 mg of Vitamin B6 hydrochloride, and 100 mg of thiamine hydrochloride in 500 ml of ultrapure water using a magnetic stirrer. Carefully mix aliquot in 50 ml tubes and store it at 20 degrees Celsius. To prepare riboflavin solution, dissolve 100 mg of riboflavin in 500 mg of ultrapure water using a magnetic stirrer.

Carefully mix aliquot in 50 ml tubes and store at 20 degrees Celsius. To prepare one molar calcium dichloride dissolve 7.35 grams of calcium chloride dihydrate in 50 ml of ultrapure water using a magnetic stirrer. For Minimum Essential Medium preparation dissolve 400 mg of potassium chloride 6, 800 mg of sodum chloride, 2, 200 mg of sodium bicarbonate, 158 mg of sodium phosphate monobasic dihydrate, 7, 000 mg of D-glucose, and 200 mg of magnesium sulfate heptahydrate, and 950 ml of ultrapure water, using a magnetic stirrer.

Next, use a 1 ml pipette with a constant agitation on a magnetic stirrer to add 1.8 ml of 1 molar calcium dichloride to the MEM in a drop by drop manner. Then add hydrochloric acid to adjust the pH of the MEM to pH 7.25. Then, add 5 ml of 200x vitamin mix and 200 microliters of riboflavin solution to the MEM.

Adjust the volume the volume of the solution to 1000 ml with ultrapure water. Filter the solution using a 0.22 micron filter system and store it at 4 degrees Celsius. To prepare 10x DNase-1 stock solution dissolve 100 mg of Dnase-1 in 12.5 ml of HBSS.

Filter through a 0.22 micron filter, aliquot in 1.5 ml tubes, and store the tubes at 20 degrees Celsius. For Ara-C stock solution preparation dissolve 25 mg of Ara-C in 8.93 ml of ultrapure water. Filter through a 0.22 micron filter, aliquot in 1.5ml tubes, and store at 20 degrees Celsius.

To prepare the Plating Medium, mix 1 ml of MEM Amino Acid Solution, 750 microliters of 1 molar HEPES, 1 ml of B27, 125 microliters of 200 millimolar glutamine, 250 microliters of penicillin-streptomycin, 2.5 ml of Fetal Bovine Serum, and 44.375 ml of MEM in a 50 ml tube. To prepare the stop medium, mix 1 ml of MEM amino acid solution, 750 microliters of 1 molar HEPES, 5 ml of FBS, and 43.25 ml of MEM in a 50 ml tube. To prepare HBSS, supplemented with 15 millimolar HEPES, mix 49.25 ml of HBSS, and 750 microliters of 1 molar HEPES.

For poly-l-lysine coated dishes preparation, coat 35 mm plastic cell culture dishes with 0.2 mg per ml of poly-l-lysine hydrobromide for one day at 25 degrees Celsius. Next, wash the dishes with 2 ml of ultrapure water three times and incubate them with 1.5 ml of Stop Medium at 25 degrees Celsius until use. Incubate the dissected hippocampi in a mixture containing 500 microliters each of 2.5%trypsin and 10X Dnase-1 in HBSS supplemented with 15 millimolar HEPES, pH 7.2, at 37 degrees Celsius for 15 minutes with agitation every 3 minutes.

Then, transfer the hippocampi to 10 ml of the Stop Medium and incubate at 4 degrees Celsius for 5 minutes to inactivate trypsin. Next, incubate the dissected hippocampi in 10 ml of fresh stop medium at 4 degrees Celsius for 5 minutes. Move hippocampi into 10 ml of fresh stop medium and incubate at 4 degrees Celsius for another 5 minutes.

Then, move the hippocampi into 900 microliters of the stop medium and 100 microliters of 10X Dnase-1 in a 15 ml tube. Use a 1 ml pipette to dissociate the hippocampi into isolated neurons by pipetting 20 times. Add 9 ml of plating medium, and filter through a 70 micron cell strainer, into a a 50 ml tube.

Count the number of cells using a hemocytometer and a adjust to 3.5 times 10 to the fourth cells per milliliter in plating medium. Next, aspirate the stop medium from the poly-l-lisine coated dishes. Plate 2 ml per dish of the cells on the coated dishes and incubate under 5%carbon dioxide at 37 degrees Celsius for 60-64 hours.

After the incubation, add 2 microliters of Ara-C stock solution to the neurons and shake the dish slowly. Keep the culture dishes in a humidified box without changing the culture medium under 5%carbon dioxide at 37 degrees Celsius. Purify the dendritic filopodia rich fraction after 13 days in vitro by first adding three million magnetic polystyrene microbeads per dish to 20 dishes containing the cultured neurons.

After one day, wash the neurons in 1 ml of PBS with agiation three times to remove the medium and unbound microbeads. After removing PBS, lyse the neurons with 500 microliters per dish of the lysis buffer. Next, collect the lysate with a cell scraper and transfer the lysate into 10 low protein binding microtubes.

Set the tubes on a magnet separater and wait for one minute. Collect the supernatant and use it as the unbound fraction for silver staining and Western blot analysis. Next, transfer the beads to a new low protein binding microtube and set on a magnetic separater for one minute.

Completely remove the supernatant, and add 500 microliters of the lysis buffer. Wash the beads using a Vortex mixer for 15 seconds. Repeat the washing of the beads 10 times and remove the supernatant.

Elute proteins down to the beads by the addition of 50 microliters of 1X SDS sample buffer and boil the tube at 98 degrees Celsius for 5 minutes. Centrifuge the tube at 860xG 3000 RPM for 10 seconds and set the tube on a magnetic separater for one minute. Collect the supernatant and use it as the bound fraction.

Measure concentrations of the unbound and bound protein fractions by the BCA protein assay. Visualize protien solutions with bromophenyl blue and adjust the concentration to 5 nanograms per microliter for SDS page. Separate the bound and unbound fractions by SDS page using a 5-20%gradient gel.

Silver stain the gel. Finally, Western blot using appropriate primary and secondary antibodies according to the manuscript. In cultured hippocampal neurons, TLCN was abundantly localized to the dendritic filopodia, shaft, and soma and co-localized with F-actin.

The encoded beads were mainly bound to dendrites and induce the formation of phagocytic cups by accumulation of TLCN and F-actin on neuronal dendrites. Thorecense images showed that phagocytic cup formation was crucially dependent on the presence of TLCN in dendrites. The phagocytic cups were only formed on wild type hippocampal neurons, but not on TLCN defficient hippocampal neurons.

SDS page results showed that the protein band patterns were almost the same for the unbound and bound fractions but the intensities at at 50 and 70 kilodalton, in the bound fraction were lower than those in the unbound fraction. However, the band intensity was not obviously different between the unbound and bound fractions prepared from TLCN defficient culture hippocampal neurons. Western blot analysis of the unbound and bound fractions showed that TLCN and VN were mainly detected in the bound fraction.

Actin, Ezrin, G alpha q, PLC beta 1, MAP-2, and Spectrin, were detected in both the bound and unbound fractions. Moesin, PSD-95, alpha-Actinin, and alpha-Tubulin were detected in the unbound fraction. If coating protein of the microbeads change, this procedure can be applied to identify assay interaction.

This technique could identify the protein working on artificial substances. So we believe that mechanism artificial genesis can be quantified. We believe that new protein associated with mental disorder can be identified from the dendritic filopodia-rich fraction.

And can be extended to the therapy.

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Journal (Neuroscience)

Journal (Neuroscience)

Purification of the Dendritic Filopodia-rich Fraction

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