Automacs cell sorter

The Institutional Animal Care and Use Committee (IACUC) at the Medical University of South Carolina approved all experimental protocols. Male and female, age-matched mice (8-12 week age range; 5 per group), on a mixed C57/129 background, were obtained for peripheral blood (PB) by cardiac puncture and primary bone marrow cells (BM) as previously described (17 (link)). PB samples were run on a Scil Vet ABC complete blood counter (CBC) to determine distribution of lymphocytes, granulocytes and monocytes (Scil Vet, Gurnee, IL). Cells (PB and BM) were immunophenotyped using makers against T cells (CD4), B cells CD45R (B220), granulocytes (Gr1) and monocytes (CD11b) and CD115 (Colony-Stimulating Factor 1 Receptor). All antibodies were obtained from Miltenyi Biotech (Auburn, CA) and attached to fluorophores. Following immuophenotyping analysis, Gr1⁺ granulocytes (Ly6G⁺) were obtained from whole BM using antibodies conjugated to magnetic beads. Osteoclast progenitors were isolated from both male and female BM cells as previously described (17 (link)). Briefly, using the AutoMACS cell sorter (Miltenyi Biotec Inc., San Diego, CA, USA), BM cells were separated by expression of integrin CD11b into CD11b^hi, CD11b^lo and CD11b⁻ fractions, were the CD11b^lo/− fractions are negative for lineage markers including CD3, CD45R, Gr1, CD11c and positive for CD115.

Mice were exposed to 850 cGy total body irradiation with the use of a [¹³⁷Cs] source 8 hours before HCT. Recipients were injected i.v. with either DBA/2 CD25⁺-cell depleted spleen cells and whole bone marrow (BM) cells or C57BL/6 whole spleen cell and T and B cell-depleted BM (TBCD-BM). CD25 depletion in the spleen, and T and B cell depletion in the BM was accomplished using biotin-conjugated anti-CD25 (spleen) and biotin-conjugated anti-CD3, anti-CD4, anti-CD8, anti-B220 and anti-CD19 mAb (BM) and anti-biotin micromagnetic beads, followed by passage through an autoMACS cell sorter (Miltenyi Biotec). The purity of depletion was >99%. The assessment and scoring of clinical cutaneous GVHD was described previously [21 , 22 , 65 (link)]. Assessment and scoring of total GVHD was as follows: hair loss (0–2), proteinuria (0–2), activity (0–2), posture (0–2) body weight loss (if no proteinuria occurred) (0–2).

Approval for collection of all primary samples was obtained from the Institutional Review Board of Northwestern University. Patients provided written informed consent in all cases at time of enrollment in accordance with the Declaration of Helsinki. An AutoMacs cell sorter (Miltenyi Biotec) was used to purify CD138⁺ cells from multiple myeloma patient bone marrow aspirate as described previously (3 (link)). Normal peripheral blood mononuclear cells (PBMC) were harvested by layering whole blood over a Ficoll histopaque (Sigma) gradient and subject to standard buffy coat purification.