The procedure described below details how to perform intracranial tumor cell injection in mice. This is a major surgery and all procedures must be approved by IACUC or other animal use committee.
Prepare cells for injection:
In the tissue culture room, typsinize cultured cells.
Wash cells with sterile PBS, and then resuspend in sterile PBS to an adjusted final volume of at least 100,000 cells per 3ul injection.
Cells should be transported to the injection area on ice.
Assemble the stereotaxic device according to manufacturer’s instructions.
Lay out the drill and drill bit.
Turn on the heating pad and monitor its temperature with a thermometer.
Load Hamilton syringe with cells:
Prepare the Hamilton syringe by rising in 70% Ethanol and then sterile PBS.
Gently resuspend trypsinized cells in PBS.
Draw cells to 3uL in the Hamilton syringe, carefully avoiding bubbles.
Position the syringe in the stereotactic device, and tighten the screw to lock the syringe in the correct position.
Anesthetize mouse by intraperitoneal injection of Ketamine 100 mg/kg. Confirm anesthetic effectiveness by toe pinch.
Wipe the mouse’s head 3 times with sterile cotton swab dipped in betadine, then swab with an alchohol wipe.
Protect the mouse’s eyes by coating with sterile ocular lubricant.
Make a 1.5 cm sagittal incision in the mouse's scalp from the front to the back.
Position the mouse in the stereotactic apparatus: first displace the tongue to the side with the wooden end of a sterile cotton swab, then position the front teeth in the tooth holder, next tighten the nose holder until firm, and finally place both ears in the ear holders. Once the mouse is in the correct position, tighten the ear holders until firm.
Wipe the skull with a sterile cotton swab to remove the shiny membranes and to aid idenification of the bregma suture.
Drill a hole 2 mm right and 1 mm anterior to the bregma suture.
Lower the Hamilton syringe needle to the edge of the hole. Lower the needle 3.5 mm, into the brain. Raise the needle 1mm and wait 1 minute. Inject 1 uL of cells, raise the needle 1 more mm, and wait 1 minute. Inject 1 uL, raise 1 mm, and wait 1 minute. Then inject the final 1 uL.
Completely remove the needle from the mouse’s brain.
Remove the mouse from the stereotaxic device.
Close the skin incision by holding the edges with forceps and applying one clip.
Place the mouse on the heating pad until the effects of anesthetic wear off, at which time the mouse can be returned to its cage.
Clean the Hamilton syringe with Hamilton cleaning solution, then 70% Ethanol, then sterile water.
Administer analgesic twice a day for the first 3 days after surgery.
Remove sutures after 10 days.
Monitor mice daily for signs of neurologic compromise.