1. Depletion of CD45+ Cells

2. Bone Marrow Transplant Aims Will replacing stem cells rescue premature age-related bone loss? Which stem cells contribute to the rescue? –Separate hematopoietic and osteogenic cells so can transplant separately

3. Mouse Model of Aging Single mutants: Terc -/- or Wrn -/- Double mutant: Wrn -/- Terc -/- Mononuclear cell isolation CD45 depletion

4. Long Bones Femurs Tibiae Humeri

5. “Spine” Hip Bones Spine

6. Mononuclear Cell Isolation Long Bones Spine Ficoll Flush out marrow Crush bones

7. Procedure Incubate w/ biotinylated  -CD45 Incubate w/ streptavidin- conjugated Dynabeads Magnetic separation Magnetic separation + column Repeat

8. Magnetic Separation Dynabead CD45+ Cell Trash Save for further purification

9. Pass cells through column for separation of beads Cells plated, fixed after 3 days CD45- antibody, streptavidin- conjugated cy-3, dapi

10. DAPI CD45+ Background flourescence DAPI CD45 Depleted Bone Marrow

11. Untreated Bone Marrow DAPI Untreated Bone Marrow CD45+ Merged

12. Already depleted from long bones of 4 WT mice, fixed after 3 days Control- cytospin BM onto slide, fixed CD45- antibody, streptavidin-conjugated cy-3, DAPI, and sca1 Next