Creation of bone marrow chimeras

 

  1. Lethally irradiate recipient mice (6-8 weeks old) the day before you plan to do the bone marrow transfers. Lethal irradiation of C57BL/6 mice is done by 2 doses of 500 rads, 24 hours apart. Allow at least 6 hours (and up to 24 hours) after the second radiation dose before transferring the bone marrow.
  2. Kill donor mouse by CO2, remove femurs/tibias and clean off muscle. Cut off the top end of each bone.
  3. Puncture hole in 0.5 ml thin wall tube with 20 to 22 gauge needle (gauge size is important). Place bone facing down in the 0.5 ml tube, and then place tube into a 1.5 ml tube containing 100 ul of RPMI. Spin down for 1.5 min at 4000 G in a microfuge.
  4. Recover cells, centrifuge, and resuspend in 10 mls of RBC lysis buffer. Spin down immediately for 5 min.
  5. Remove supernatant and resuspend in 500 ul Sterile PBS
  6. Count cells, make the concentration of 4 x 107 cells/mL in sterile PBS. Expect 3-4 x107 cells per mouse.
  7. Transfer 50 μL (2 x 106 cells) i.v. into each irradiated recipient.
  8. Maintain recipient mice on antibiotics (water or diet via the core facility). After 6 weeks, the animals are ready for tumor implantation.