Cd19 microbeads isolation kit

Primary samples from patients suffering from MCL and CLL were collected at the time of diagnosis with informed consent from San Gerardo Hospital (HSG, Monza, Italy), according to the IRB‐approved protocol (HSG IRB #926, code: CD24.DEM) accepted by Local Ethical Committee. Tumour population was obtained from patients' blood that was diluted with PBS (dilution 1:2) and separated on Lympholyte‐H from Cedarlane to get PBMCs. Tumour cells were then isolated from PBMCs by CD19 Microbeads isolation kit from Miltenyi Biotec according to the manufacturer's protocol. Isolated cancer cells were then checked for purity by flow cytometry (CD45+/CD19+ cells >90% by flow cytometry). Patients‐derived monocytes were selected with the same protocol used for donors' monocytes. Leftover samples from healthy donors for PBMCs and monocytes collection were obtained after plateletpheresis in Transfusion Medicine Unit (HSG, Monza, Italy), after signing informed consent. All the procedures outlined in the study were conducted in accordance with the ethical principles of Helsinki Declaration.

The following anti-mouse fluorescently labeled antibodies were used: CD19 (clone 1D3), CD11b (clone M1/70), CD5 (clone 53-7.3), CD80 (clone 16-1OA1), MHCII (clone 2G9), CD86 (clone GL1) (BD Biosciences, Germany), and CD23 (clone B3B4) (eBiosciences, Germany). Mitomycin-c was obtained from Sigma-Aldrich, Germany. CD19 MicroBeads isolation kit, CD5 Microbeads isolation kit, and regulatory T cells isolation kit were obtained from Miltenyi Biotec, Germany. Anti-mouse IL4 and anti-mouse IFNγ were from BD, Biosciences, Germany. TGFβ was purchased from R&D System, Germany. IL23 and IL6 were obtained from eBiosciences, Germany. Cytometric bead array (CBA) Mouse Th1/Th2/Th17 Cytokine Kit was obtained from BD, Biosciences, Germany.