5% dimethyl sulfoxide (DMSO) and stored in liquid nitrogen. BECs were separated from adherent cells using CD326 (EpCAM) conjugated MicroBeads (Miltenyi Biotec) specific for epithelial cells. Cells were then resuspended in media consisting of a 1:1 mixture of Ham’s

F12 and Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 5% fetal calf serum PD0325901 mouse (FCS), epithelial growth factor (10 ng/mL), cholera toxin (10 ng/mL), hydrocortisone (0.4 μg/mL), tri-iodothyronine (1.3 μg/L), transferrin (5 μg/mL), insulin (5 μg/mL), adenine (24.3 μg/mL), and 10 ng/mL hepatocyte growth factor (R&D systems, Minneapolis, MN) and cultured.7 The purity of the cells was verified by immunohistochemical examination of an aliquot of these cells for the expression of cytokeratins 7 and 19 using appropriate antibodies (Dako, Glostrup, Denmark) and only cultures that were >90% positive

for these cytokeratins and >95% viable (as determined by trypan blue) were used for the studies reported herein. The cultures used in the studies herein were between four to six passages to exclude the possibility for potential loss of phenotype after prolonged in vitro culture. As reported,8 the T cells used for the studies were isolated from LMC using a Pan T cell isolation kit II (Miltenyi Biotec).8 Similarly the highly enriched population of Mo and NK cells used were purified using Mo and NK cell isolation kits, respectively (Miltenyi Biotec).8 The purity of the CD3+ T cells, Mo, and NK cells used were >90% as determined by flow cytometric selleck compound analysis of an aliquot from each isolation. In efforts to

ensure learn more the purity of the cell population being studied, the population of T cells, Mo, or NK cells were each harvested separately. In addition, the same assay was performed following depletion of each of the three cell lineages from LMCs in efforts to confirm that the data obtained were indeed the function of the lineage being studied. The mDCs (BDCA-1+), pDC (BDCA-2+), and NKT cells were isolated using the mDC, pDC, and NKT cell isolation kits (Miltenyi Biotec), respectively, which included two magnetic separation steps. The purity of BDCA-1+ mDCs and the CD3+ CD56+ NKT cells were each >80% as determined by flow cytometric analysis of an aliquot of the cell preparation used for the study. An enriched population of mDC and NKT cells were harvested separately and, once again, the same assay was performed following depletion of the specific cell population in efforts to confirm that the function identified was due to the specific cell lineage being studied. The cytotoxic activity of LMC was assessed using an 8-hour 51Cr release assay using autologous BEC as target cells.