BMMs and T cells were purified from the BM and spleen by positive immunoselection using MACS Microbeads (Miltenyi Biotech,) coupled to anti-CD11b, anti CD90, or anti CD4 and anti CD8 antibodies, as described [11], [19].
Isolation of CD14+ cells was performed with magnetic beads (CD14 Microbeads, Miltenyi Biotec, Madrid, Spain) by positive immunoselection using the autoMACS Separator (Miltenyi).
PBMC were separated into CD4+ and CD8+ T cells by positive immunoselection, using magnetic beads (MiniMacs, Miltenyi Biotec, Auburn, CA, USA) according to the manufacturer's recommendations.
Dendritic cells were generated from CD14+ monocyte precursors purified from freshly isolated PBMC by positive immunoselection using an anti-CD14 mAb coupled onto magnetic microbeads (Miltenyi Biotech, Auburn, CA, USA) under the manufacturer's protocol.
After discarding the non adherent cells, adherent BMMs were eliminated by positive immunoselection by MACS Microbeads (Miltenyi Biotec, Auburn, CA) coupled to anti-CD11c antibody.
Alternatively, CD4+ T cells were purified by positive immunoselection with anti-CD4 microbeads (>90% CD4+ T cells; Miltenyi Biotec, GmbH, Bergisch Gladbach, Germany).
This was followed by immunoselection using mouse anti-human PSMA antibody (clone K0142-3, MBL International, Woburn, MA, USA) followed by collection of PSMA-expressing cells using sheep anti-mouse Ig-coated paramagnetic beads (Life Technologies, Paisley, UK).
CD4+, CD4+, CD45A+, CD8 and CD8+CD45A+ T-cell subsets were isolated from PBMC by either positive (CD4) or negative (CD8) immunoselection, using mag-netic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) (Fig. S1).
After washing, CD11c+ cells were purified by positive selection using MACS separation columns (Miltenyi).
In these experiments, HMCLs or CD138+ cells were removed using magnetic cell-sorting separator, whereas B lymphocytes were removed using a positive immunoselection with anti-CD19 mAb (Dynal, Lake Success, NY, USA), and the BMSCs were subjected to RNA or protein extraction.
MSCs isolated directly from bone marrow are positive for CD34, the hallmark antigen for positive immunoselection of the hematopoietic stem cell, but lose this antigen upon in vitro culture.
