Immunophenotyping of Transplanted Cells

Mice were euthanized at 19 weeks post-transplantation and whole femurs and spleens were removed. Femurs were flushed with Dulbecco’s phosphate buffer saline to remove marrow. Single cell suspensions of bone marrow and spleen cells were created and processed as previously described,^{35 (link)} except that commercially available red blood cell lysis buffer was used as described above. Cells were counted and 2 × 10⁵ cells/staining reaction were distributed to the wells of a 96-well round bottom tissue culture plate. Cells were stained with antibodies as previously described,^{35 (link)} except that Phycoerythrin (PE) conjugated anti-human CD3 (Clone UCHT1), PE conjugated anti-human CD11b (Clone ICRF-44), PE conjugated anti-human CD34 (Clone 581), and PE conjugated mouse IgG1, k isotype control (Clone MOPC-21) antibodies (BD Biosciences) were also used. Cells were washed and analyzed by flow cytometry. Statistical significance was determined by performing t-tests.