The Lonza cells were thawed and washed with PBS and directly used. Here are the details:
Thaw cryopreserved cord blood huCD34+HSCs (1×106 CB-CD34+cells per vial for 8 to 10 mice) in a 37°Cwater bath until a sliver of ice remains. Then, transfer cells
to a 15-ml polypropylene tube in a class II BSC and wash twice in 10 ml of 1× PBS at room temperature, with centrifugation for 5 min at 300×g. Resuspend cells in 0.5
to 1 ml RPMI-1640 medium and count viable cells either manually, using a hematocytometer, trypan blue, and a light microscope, or with an automated cell counter. 3. Adjust concentration of huCD34+ HSCs by resuspension to 6 × 105 cells/ml in RPMI-1460 and store cells briefly on ice prior to injection. Using a 1-cc tuberculin syringe with a 25-G × ?-in. needle, inject 0.2 ml (1.2 × 105 HSCs) into lateral tail vein of each irradiated mouse by intravenous (IV) injection.