Cytotoxicity and Immune Cell Migration Assays

For cytotoxicity assay, lactate dehydrogenase (LDH) activity was measured using a Cytotoxicity detection kit (Roche Diagnostics) according to manufacturer’s protocol. For pinocytosis assay cells were incubated with 0.25 mg/ml FITC-Dextran (Sigma-Aldrich) at 37°C for 20 minutes and subsequently analysed using flow cytometry. TLR3 agonist Polyinosinic-polycytidylic acid sodium salt (Poly(I:C)) (20 μg/ml) (Sigma-Aldrich) was added to Mo-M cultures on day 5. For migration assay of T cells, T_regs and B cells, a SPLInsert™ Hanging 3μm pore size (SPL Life Sciences) migration chamber was used. 2x10⁵ isolated T cells, T_regs or B cells were allowed to migrate towards conditioned media from M2 or M2/IFN treated CD169⁺ Mo-M for 18h h, or serum as positive control, with subsequent 4% PFA fixation of transmigrated cells and subsequent Cytospin with H/E staining was performed prior to counting. For cell lines, MDA-MB-231 (ATCC) and SUM159 (a kind gift from Professor S. Ethier (27 (link)) and bought from BioIVT, NY, US) TNBC breast cancer cells were used.