Protocol: Pump Implantation

Species:  Mouse.

Depending on purpose of experiment:  The mice are sublethally irradiated with 320 rads either the day before or same day as the injection. The experimental procedure involves injecting human colon cancer cell suspensions under the renal capsule of  NOD/SCID mice.

Steps:

• The mice are handled using sterile technique and anesthetized using inhalational anesthesia, isoflurane. The mice are placed on a heating pad during the procedure.

• A clipper is used to shave the abdomen, which is then prepped sequentially with: (1) iodine based solution and (2) 70% ethanol solution.

• The mouse is placed on it’s side (left side up, we always inject the kidney on the left side (same side as the spleen)). I make a 1 cm (approximately) flank incision with scissors, just below the costal margin on the left side.

• Deliver the kidney into the wound (this is difficult to explain but is very clear once you see it). You will need two pairs of forceps.

• Utilizing a tuberculin syringe inject the cell suspension under the renal capsule. I load all the syringes at the lab and bring them to the colony on ice. The cells are mixed 1:1 with media and matrigel. If the matrigel is not kept on ice you may have a hard time emptying the syringe. I aim to suspend the cells in 10ul of media. I have my matrigel aliquoted and frozen at -20C (it is not diluted). I thaw an aliquot and draw it up into a 1ml syringe (no needle). I then insert a small amount of the matrigel into the back of  a tuberculin syringe (remove the plunger). It is difficult to be exact on the amount of matrigel but I aim for it to be 25-50ul (closer to 25 is better), the best way to estimate the amount is looking on the side of the syringe. I then use a pipette to inject my 10ul of cells into the middle of the matrigel. Once that is done I re-insert the plunger into the syringe and push the mixture to the end of the syringe.

• Deliver the kidney back into the abdomen, close the abdominal wall in one layer, take peritoneum, muscle, and skin in that layer. Use a running 4.0 vicryl suture.

• After the procedure is completed, subcutaneously administer 1 cc of sterile saline and .02cc Banamine (analgesic).

• During the initial recovery period (20-30min), utilize a heating pad and/or light to ensure that the mice are not rendered hypothermic.