Slan (M-DC8)+ Monocyte Isolation Kit, human

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		Slan+ monocytes or CD14+ monocytes (2.5 × 104) were thereafter dispensed into U‐bottom 96‐well plates (Corning, Corning, NY, USA), and cultured for 24 h in RPMI 1640 medium containing 10% FBS, in the absence or in the presence of 20% cell‐free supernatants from SW480, SW620, and MCF7 cell lines.

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Slan+ monocytes or CD14+ monocytes (2.5 × 104) were thereafter dispensed into U‐bottom 96‐well plates (Corning, Corning, NY, USA), and cultured for 24 h in RPMI 1640 medium containing 10% FBS, in the absence or in the presence of 20% cell‐free supernatants from SW480, SW620, and MCF7 cell lines.

Publication protocol

A human breast carcinoma cell line (MCF7; from ATTC, Manassas, VA, USA) and 2 colon‐adenocarcinoma cell lines (SW480, a primary tumor, and SW620, its matched lymph node metastasis; both from ICLC, National Institute for Cancer Research, Genoa, Italy) were used in this study.18 Cell lines were cultured in DMEM (MCF7 and SW620), or RPMI medium (SW480), supplemented with 10% endotoxin‐free FBS. Cell‐free supernatants were collected after ∼3 d of culture, a time point at which cell lines reached ∼70–80% confluence. PBMCs were isolated by density gradient centrifugation of buffy coats from healthy donors (on Ficoll‐Paque; GE Healthcare, Little Chalfont, United Kingdom), under endotoxin‐free conditions. Slan+ monocytes and CD14+ monocytes were then purified with, respectively, the slan (M‐DC8)+ Monocyte Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) (>96% purity) and the CD14+ MicroBeads (Miltenyi Biotec) (>98% pure).18 Slan+ monocytes or CD14+ monocytes (2.5 × 104) were thereafter dispensed into U‐bottom 96‐well plates (Corning, Corning, NY, USA), and cultured for 24 h in RPMI 1640 medium containing 10% FBS, in the absence or in the presence of 20% cell‐free supernatants from SW480, SW620, and MCF7 cell lines. After various times (see Fig. 3), monocytes were detached by incubation for 15 min on ice with PBS containing 2% FBS and 2 mM EDTA and then stained for 15 min at room temperature with anti‐slan (M‐DC8)‐FITC mAbs (from Miltenyi Biotec). Phenotypic analysis of monocytes cultured under the various experimental conditions was performed on live cells, identified by the Vybrant DyeCycle Violet kit (Thermo Fisher Scientific).18 The fluorescence of samples was measured by an 8‐color MACSQuant Analyzer (Miltenyi Biotec), and data analysis was performed by FlowJo software Version 10.120 (Ashland, OR, USA). The mean fluorescence intensity (MFI) relative to the slan Ag was obtained by subtracting the fluorescence intensity of the correspondent isotype control anti‐IgM‐FITC.

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