Encyclopedia of Experiments: Immunology
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Transcript
To begin, insert the blunt-end needles to the appropriate position within the device mold and cast approximately 20g of polydimethylsiloxane or PDMS mix for one set of the device and lid. Place the molds in a vacuum chamber for 30 minutes to remove air bubbles from the PDMS mix, then, incubate the molds at 55 degrees Celsius overnight to complete PDMS polymerization.
When the PDMS is set, pull out the needles from the mold and carefully release the culture device and lid from the plastic molds. Remove PDMS residues from the well outline using a surgical blade.
Attach PDMS device in the device cover onto a cover glass of 75 by 50-millimeter microslides using non-toxic silicone adhesive, and leave the parts to set overnight. Apply the glue to the smooth side of the device. Insert 12 22-gauge needles for the lumen and 12 18-gauge needles for the well. Fix all the needles in place using silicone and let it set overnight.
Purge the input syringes, and make sure that the well medium flows out from all tubes into a waste glass. Then, purge the input syringes, and make sure that the stimulations flow out of all the tubes into a waste glass, taking care to not contaminate the different stimulations.
After sacrificing the mouse, use sharp scissors and forceps to dissect it, and take out the digestive tract from the stomach to the anus by cutting all the fat and connective tissues. Cut the colon and place it on a new plate. Minimize contact while holding the tissue gently and only at the edges. Perform the colon flush under the dissection microscope.
Gently flush the colon content with sterile IMDM with the prepared 10-milliliter syringe as described in the text. After removing the feces from the intestinal tissue, place the colon in a new 6-well plate filled with 0.5 milliliters of sterile IMDM. Next, take the colon tissue and carefully connect it to the 22-gauge needle, making a tight tie with the two threads.
Maintain the correct orientation of the colon to the lumen flow such that the proximal and distal is equal to input and output respectively. Repeat colon flushing and needle tying for all the tissues. Connect the input and output tubes to the device, then begin the experiment by starting the pumps at the desired rates.
