To begin, remove the cryovial containing cord blood CD34+cells from the freezer. Thaw the cells in a water bath before transferring them to a 50 milliliter tube. While shaking the tube dropwise, add 10 times the cells volume of prewarmed IMDM containing 50%FBS and 0.1 milligram per milliliter DNase to the tube.
Centrifuge the cell suspension at 500 G for five minutes. Resuspend the pellet in 20 to 40 microliters of PBS containing penicillin and streptomycin. Store the cells on ice until use.
Prepare a three milliliter syringe with a 27-gauge half-inch needle. Prepare a 0.5 milliliter insulin syringe with a 29-gauge 12.7 millimeter needle containing cord blood CD34+cell suspension. Next, prepare a one milliliter insulin syringe with a 29-gauge 0.5-inch needle with 100 microliters of buprenorphine.
Transfer the anesthetized mouse to a nose cone and perform a toe pinch to confirm its readiness for the injection. Lay the mouse on its back and flex its hind legs. To secure the hind legs, place the thumb of the nondominant hand on the foot, the middle finger on the hip, and the index finger on the outside of the femur.
Gently pluck the hair from around the kneecap and wipe the area with an alcohol swab. Now using a three milliliter syringe with a 27-gauge half-inch needle, gently drill a hole through the skin towards the femur. Once the needle is in the bone, rotate it clockwise until the whole needle is in the bone.
Remove the needle using an anti-clockwise rotation until halfway out. Wipe around the needle with an alcohol swab and continue to turn the needle anti-clockwise for complete removal. Next, insert the 0.5 milliliter insulin syringe containing cell suspension into the femoral shaft.
Once the needle is in the bone, release the grip on the leg and gently inject 25 microliters of suspension into the femur. Finally, inject the buprenorphine subcutaneously and return the mouse to its home cage, allowing it to recover from anesthesia.
