TITLE: UABMC - PDX Passage Protocol
AUTHORS: Christopher Willey

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							Select 1 to 2 mice that have tumors that measure no more than 8-10mm in greatest diameter, are not ulcerated and appear to have grown in a consistent fashion.
(This must be done aseptically using sterile surgical techniques
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Euthanize one mouse at a time with CO2 asphyxiation in designated inhalation chamber attached via a two-stage regulator to a CO2 tank.  Perform cervical dislocation or bilateral thoracotomy to ensure the mouse is dead.
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Spray the mouse with 3% chlorhexidine sanitizing solution (over the sink) and transfer mouse to a sterile convertor cloth.  Sanitize the skin around the tumor with betadine scrub solution.  Wait at least 2 mins before proceeding.
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Use sterile forceps and one scalpel with a #11 blade to produce an incision in the skin forming a half-circle about 5mm away from the tumor on the ventral (lower) side of the tumor.
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Use the forceps to lift the edge of the skin and use a new sterile scalpel with #11 blade to carefully dissect under the tumor freeing it from the underlying muscle tissues. Blunt dissection works best, where possible.  Use your ring finger of the hand holding the forceps to push the tumor forward during this dissection.
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After you have dissected the tumor free from the body of the mouse, continue to dissect the tumor from the overlying skin.
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Open one of the glass Petri dishes and set the cover aside.  Continue the dissection of the tumor such that it “drops” into the open dish. Cover the Dish and transfer to a laminar flow biological safety cabinet.
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Dice tumor into large pieces (chunks) and dissect out any necrotic (dead) tissue. Necrosis can be white, yellowish or dark red and bloody.  Transfer healthy-looking tumor tissue chunks to a new glass Petri dish, if necessary.  Place Petri dish on an ice bath until all tumor chunks have been harvested and processed to this point.
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Place the carcass in the black plastic sack and proceed to euthanize the next mouse and repeat the process.  Ensure that the carcass bag is well tied and disposed in the Morgue in Animal Resource Program space.
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Rinse tissue chunks a couple of times with 5-10 ml each of sterile PBS; aspirate as much rinse solution as possible each time from Petri dish.
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Mince rinsed PDX tissue chunks finely using 2 #11 scalpel blades in a scissor-like action until the pieces are small enough to fit through the bore of a 10ml pipet.
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Add  PBS to the petri dish with the minced tissue.
200 µl
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Using a sterile 1ml slip-tip tuberculin syringe (without needle attached), draw up the tissue
We typically implant 100 – 250ul of tissue per mouse. The hub of the needle will have about 100ul of “dead” space, so draw up enough tissue to compensate for this loss.
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Fit a 16 gauge 1½ inch needle on the syringe tip and expel any air bubbles and excess liquid. Place the syringe on ice.
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Anesthetize the mice using ketamine/xylazine by intraperitoneal injection or isoflurane by inhalation. (Refer to Anesthesia SOP#_______)
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Place the mice on a sterile convertor towel on their side, right flank facing up. Apply Chlorhexidine scrub with a sterile cotton-tipped applicator to the skin on the Right flank, wait a couple of minutes, then wipe with sterile isopropanol wipe to remove excess chlorhexidine.  Allow alcohol to dry.
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Re-sanitize your gloves.  Pull the skin of the right flank between your fingers, smoothing it out and lift gently but tightly.
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Using your middle finger as a guide underneath the skin fold, slowly insert the needle just under the skin. The needle should be visible through the skin. Slowly push the desired amount of tissue chunks into the area on the mouse’s side between the front and rear legs. Draw the needle out slowly and pinch the skin together with a pair of semi-curved forceps as the needle retracts. Place a small drop of tissue glue on the needle entry wound.
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Place the mice back in a clean sterile cage; lay the mice on their left side in the cage). Place the cage half on, half off of a heating pad set to low () until the mice are awake and able to move around normally.
84 °F