Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

The overall goal of this procedure is to track the retrograde axonal transport of brain-derived neurotrophic factor or BDNF by live imaging. This is accomplished by first assembling microfluidic chambers on polylysine coated cover slips. In the second step dissociated rat E 18 hippocampal neurons are dissected and plated in the cell body compartment of the microfluidic chamber.

Next, the neurons are cultured to allow extension of the axons across the micro grooves into the axonal compartment of the chamber. In the final step, quantum dot labeled BDNF is added to the axon compartment. Ultimately, fluorescence microscopy can be used to assess BDNF movement within the axons of the hippocampal neuron.

The main advantage of this technique over existing methods like B-D-F-E-G-I-P, or BDFM cherry labeling is that labeling BDF with quantum dot allows the light imaging of a single BDF dimers within the axons demonstrating the procedure will be, shall be. Draw a post dot from my left three, Begin by hand washing the microfluidic chambers in 1%aox, followed by three 30 minute rinses in milli Q water. Then after hand washing the chambers in 70%ethanol, lay them out to dry on param in a laminar flow hood, sterilize both sides of the chambers under UV light for 20 minutes, and then store the radiated chambers in a sterile 15 centimeter dish sealed with param at room temperature.

Next, soak 24 by 40 millimeter. Number one glass cover slips in 10%hydrochloric acid overnight on a rotator followed by three 30 minute washes with water after the last wash. Sterilize each cover slip with a dip in 100%ethanol, followed by flaming over a bunsen burner and store them dry in a sterile Petri dish at room temperature.

To coat the cover slips, lay them in a 15 centimeter culture dish and then layer each with 0.7 milliliters of 0.01%poly L lysine. After a one hour incubation in the tissue culture hood, rinse the cover slips three times again with sterile water and then dry them with a vacuum and place them in a six centimeter culture dish. Then place one of the sterilized chambers with the micro groove side facing down onto a poly L lysine coated cover slip.

Taking care not to touch the micro grooves and use a micro pipette tip to gently press down on the chamber and tightly seal the assembled microfluidic chamber. Now placed two E 17 to E 18 rat Hippo Campi into a 15 milliliter conical tube containing two milliliters of dissection buffer and rinse the tissues three times with five milliliters of dissection buffer each time. Then remove as much of the wash as possible and add 900 microliters of fresh dissection buffer to digest the tissue.

Next, add 100 microliters of 2.5%trypsin to the tissues and place the tube in a 37 degree Celsius water bath. After 10 minutes of digestion, add 100 microliters of DNA one and use a fire polished PE glass pipette to gently tri rate the tissues up and down five to 10 times immediately after the mixing. Quench the tryin with two milliliters of plating medium and allow the debris to settle to the bottom of the tube for five minutes in the tissue culture hood.

Next, carefully transfer two milliliters of supernatant to a clean sterile 15 milliliter conical tube and spin down the cells resus. Suspend the pellet in 50 microliters of plating media, and then after counting, load 15 to 20 microliters of the cell suspension into one compartment of the microfluidic chamber. Incubate the chamber for 10 minutes to allow the cells to attach to the cover slip, and then add more plating media to fill up both compartments of the chamber.

After one day of incubation, replace the plating media with maintenance media in both the cell body and the axon compartments. On day three, axons from the hippocampal neurons start to cross the micro grooves reaching the axon compartment between days five and seven. During this time, replace half of the culturing media with fresh maintenance media every 24 to 48 hours prior to live imaging of the quantum labeled BDNF Axonal Transport.

Thoroughly rinse both compartments of the microfluidic chamber with BDNF free serum free neuro basal media every 30 minutes for two hours during the BDNF depletion, incubate 50 nano molar mono biotinylated BDNF dimer with 50 nano molar quantum. Do 6 5 5 strippin conjugates in neuro basal media on ice for 60 minutes. Then replace the media in the axon compartment with 300 microliters of quantum dot labeled BDNF for four hours at 37 degrees Celsius to minimize the diffusing of quantum dot labeled BDNF into the cell body compartment.

It is very important to always maintain a higher level of media in the cell body compartment than in the axon compartment at the end of the incubation, warmup and inverted microscope, equipped with a 100 x oil objective and an environmental chamber to a constant temperature of 37 degrees Celsius and 5%carbon dioxide while the equipment is warming up. Wash off the unbound quantum dot labeled BDNF from the microfluidic chamber, and then use a set of Texas red excitation and emission cubes to visualize the quantum dot 6 5 5 signal. Finally, use a CCD camera to capture time-lapse images within the middle axons at the speed of one frame per second for a total of two minutes.

Use micro grooves with no axons that have no QD as a control for infiltration and analyze the BDNF transport using the appropriate image analysis software in time-lapse imaging of quantum dot labeled BDNF signal transport quantum dot signals are observed in most of the micro grooves with axons, while no quantum signal is seen in the micro grooves without axons indicating little to no diffusion of the quantum dot signal from the axon compartment to the cell body compartment here. An 80 micrometer long segment of a hippocampal neuron axon was fluorescently recorded for 100 seconds. A total of six.

Quantum BDNF signals were clearly noted during the imaging with three observed moving unidirectionally towards the cell body. One signal was observed to move smoothly to the cell body crossing the entire field in approximately 70 seconds. Another was observed to move in a retrograde direction for the first 20 seconds, switched to antrograde movement for 10 to 20 seconds, and then change direction again towards the cell body for the last 20 seconds.

The third event was also noted to move in a retrograde direction, but then to pause for approximately 20 seconds during the middle of the movement. In this first representative chime graph of quantum dot labeled BDNF transport, the quantum dot labeled BDNF moved quickly and smoothly towards the cell body crossing the 80 micrometer field in approximately 60 seconds. With very brief pauses in this second chronograph, the quantum BDNF traveled approximately 50 micrometers in 120 seconds in a retrograde direction with at least three segments of longer pauses.

Finally, the scatter plots measure the moving velocity, average velocity and pause time of each single quantum dot labeled BDNF. The moving speeds of the quantum dot labeled BDNF were relatively fast ranging from 0.47 to 1.97 micrometers per second with a mean of 1.06 plus or minus 0.05 micrometers per second. Because of the BDNF pausing during transport, the average velocity was much lower than the moving velocity with a mean of 0.48 plus or minus 0.03 micrometers per second with a mean duration of pauses of 15.88 plus or minus 1.30 seconds After the development.

This technique allow researchers in the field of neuroscience to explore neurotrophin and receptor trafficking in healthy and disease neurons.