Du145

Human breast carcinoma cell lines MCF-7 and HCC1143, pancreas adenocarcinoma cell line PA-TU-8902, prostate >carcinoma cell line DU145 and lung carcinoma cell line NCI-H460 were obtained from DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany).
In general RPMI 1640 medium, supplemented with 2 mM L-Glutamine and 1% Penicillin-Streptomycin and FCS (either 10% or 1% according to the assay) was used for monocyte/macrophage, NCI-H460, HCC1143 and DU145 cell culture. For MCF-7 cells we used Eagle´s MEM, supplemented with 10% FCS, 2 mM L-Glutamine, 1% Penicillin/Streptomycin and 1 mM Sodium Pyruvate and for PA-TU-8902 cells Dulbecco´s MEM High Glucose supplemented with 1 mM Sodium Pyruvate, 2 mM L-Glutamine, 1% Penicillin/Streptomycin and either 10% or 1% FCS was used (all reagents from Sigma, Buchs, Switzerland).
Cell lines were kept under standard culture conditions (37°C, 5% CO₂ and 95% humidity), tumour cell conditioned media were obtained by 24 h incubation of 90% confluent tumour cells in 10 ml of fresh culture medium in 25 cm² cell flasks. Cell free supernatants were carefully harvested, filtered through 0.5 μm filters and stored in aliquots at −80°C.

The DU-145, LNCaP, T47D and SKBR3 cell lines were purchased from DSMZ (Braunschweig, Germany), and were cultured in RPMI supplemented with 10% fetal bovine serum (FBS), at 37 °C, 5% CO₂. All media were purchased from Invitrogen (Carlsbad, USA) and all chemicals from Sigma (St. Louis, MO), unless otherwise stated.

The cell lines 5637 (DSMZ, #ACC-35), DU-145 (DSMZ, #ACC-261), KHYG-1 (DSMZ, #ACC-725), Ramos (DSMZ, #ACC-603), and ZR-75-1 (ATCC, #CRL-1500) were obtained from the DSMZ or the ATCC. All cell cultures were free of Mycoplasma and maintained in RPMI1640 medium (Biochrom, #F1215) supplemented with 10% FCS (Biochrom, #S0115) and 1% l-glutamine (Biochrom, #K0283). KHYG-1 medium additionally contained 10 ng/ml IL-2 (PeproTech, #200-02). KHYG-1 cells were stably transfected with hFcγRIIIa 158V (KHYG-1-CD16aV), cloned, and maintained in medium containing 25 nmol/l methotrexate (Sigma-Aldrich, #M8407).
Human PBMCs were isolated from leukapheresis products (Charité University Hospital Berlin) or commercially available buffy coats (DRK Berlin) of healthy donors via density gradient centrifugation using Biocoll Separating Solution (Biochrom, #L6113). In case of leukapheresis products, isolated PBMCs were stored in AIM-V medium (Life Technologies, #31035025) supplemented with 75% FCS (Biochrom, #S0115) and 10% DMSO (Sigma-Aldrich, #D2650) in liquid nitrogen for future usage. All leukapheresis donors were informed of the purpose of the donation and gave their written consent prior to the study.
PBMC subpopulations were isolated using magnetic cell separation: monocytes (Invitrogen, #11350D or Miltenyi Biotec, #130-096-537), B cells (Invitrogen, #11351D), and T cells (Invitrogen, #11344D).