Compartmentalized Primary Murine Neuron Culture Using Plastic Microfluidic Chips
Transcrição
To begin, place a sterile multicompartment chip into a Petri dish. Add 100 microliters of a precoating solution to the upper left well of the chip, and allow it to flow through the main channel into the adjoining well. Next, fill the lower left well of the chip with 100 microliters of the precoating solution.
This time, wait five minutes to allow the solution to flow through the microgrooves. Now, add 100 microliters of the precoating solution to the upper right well, and allow it to flow through the main channel into the adjoining well. After 90 seconds, fill the lower right well with 100 microliters of the precoating solution, and incubate the chip for 10 minutes.
Carefully angle the pipette tip away from the main channel and aspirate the solution from each well. Then, immediately add 150 microliters of PBS to the upper left well and wait 1.5 minutes. Next, add 150 microliters of PBS to the lower left well, and wait five minutes to allow the liquid to flow through the microgrooves.
Then, add 150 microliters of PBS to the upper right and lower right wells in that order. After 10 minutes, again, aspirate the PBS and repeat the wash steps a second time. Following the second wash aspirate the PBS the wells as before by angling the pipette tip away from the channel opening.
Then, add 100 microliters of 0.5 mgs per ml poly-D-lysine solution to the upper left well of the chip. Wait 1 and 1/2 minutes and then, fill the lower left well with 100 microliters of the poly-D-lysine solution. Repeat this process on the right half of the chip. Close the Petri dish, then place the chip in an incubator at 37 degrees Celsius for one hour.
After incubation, rinse the chip twice with PBS as previously described. Next, aspirate the PBS from the device. Immediately, add 100 microliters of cell culture media to the upper left well of the chip. After 1 and 1/2 minutes, add media to the lower left well.
Wait five minutes to allow media to flow through the microgrooves, and then repeat the filling process on the right side of the chip. Prepare a cell suspension of dissociated rat hippocampal neurons according to established protocols. Remove the majority of media in each well of the chip, but leave the channels filled.
Next immediately load 5 microliters of the cell suspension into the upper right well and another 5 microliters of cell suspension in the lower right well. When loading the cells, point the tip of the pipette towards the main channel. After loading the cells, transfer the chip to a microscope stage to make sure that the neurons are in the main channel.
After waiting 5 minutes for the cells to attach, add approximately 150 microliters of neuronal culture media to each of the upper and lower right wells. Then, add 150 microliters of media to each of the upper and lower left wells. Cover the Petri dish, and place the chip in a humidified tray in a 5% carbon dioxide 37 degrees Celsius incubator.
