Delivering Effector T Lymphocytes into Mice Carrying Human Brain Tumors
Transcrição
Ensure that the animal is adequately anesthetized by a toe pinch, and then remove fur from the surgical site between the two ears and up to the nose. Resuspend cells slowly to prevent cell clumping, and load cell suspension into syringe with great care to avoid the presence of bubbles.
Then, place the syringe into the adapted syringe pump. Disinfect the surgical site with a swab soaked in povidone-iodine 5% solution, and place a lubricating ophthalmic ointment in the mouse's eyes to prevent drying of the cornea. Then, position the anesthetized mouse on the stereotactic frame on a warm isothermal block. Appropriately position mouse's nose and teeth above the tooth bar. Tighten ear bars firmly in the mouse's ears to immobilize the head. Be careful not to damage the eardrums.
Compromise the respiration and make sure that the head is well-immobilized. Make a midline sagittal skin incision with sterile scissors along the upper part of the cranium to expose the skull. Identify the intersection of the sagittal and coronal structures to serve as landmarks for stereotactic localization, and place the syringe above this point.
Move the syringe at predetermined coordinates 2 millimeters lateral and 0.5 millimeters interior of the bregma. Using a micro-drill, make a small hole in the skull with sterile drill bit. Be careful to remain superficial in order to avoid brain injury.
Insert syringe carefully into the drilled hole. Move slowly forward the needle 3 millimeter down in the dura, and then backward 0.5 millimeters to a final depth of 2.5 millimeters prior the injection.
Run the cell injection at 2 or 3 microliters per minute and monitor the mice all along the injection time. Once injection is complete, withdraw the needle for only 1 millimeter, and keep syringe in place for an additional minute before slowly withdrawing completely the syringe to prevent any leakage from the infusion site. Remove carefully the animal from the stereotaxic frame.
Close skin with appropriate surgical suture and apply 2% lidocaine gel directly on the wound. Transfer anesthetized mouse to its respective cage above heating pad set to 37 degrees Celsius to maintain appropriate mouse body temperature until full recovery from anesthesia.
