Ficoll paque premium sterile solution

Initially, CTCs were enriched through density gradient centrifugation coupled with flow cytometry. Briefly, 4 mL of whole blood was diluted with 4 mL of phosphate-buffered saline (PBS) and gently layered into a centrifuge tube (Corning Inc, Corning, USA) containing 4 mL Ficoll-Paque Premium sterile solution (GE-Healthcare, Uppsala, Sweden). After density gradient centrifugation (1,040 × g, 20 °C, 15 min) without breaking, the interphase consisting of peripheral blood mononuclear cells and CTCs was transferred to a clean tube and washed using PBS. Red blood cell lysis was performed using ammonium-chloride-potassium (ACK) lysing buffer (Boster Biological Technology, Ltd, Wuhan, China). Subsequently, the enrichment was performed by incubating with CD45-PE-Cy5 (#555484; Becton Dickinson Pharmingen, San Diego, CA, USA) and isotype control (#555750; Becton Dickinson Pharmingen) antibody, and CTCs were negatively enriched via hematopoietic cell (CD45⁺) depletion through flow cytometry (BD FACSAria^™ IIu; Becton Dickinson). During flow cytometry, the main cell population was gated based on their forward and side scatter properties to avoid the interference of the cell debris. In the gating strategy, isotype control for each blood sample was used to gate the PE-Cy5 negative area.

Primary human monocytes were isolated from the whole blood of cancer patients before L-OHP treatment. PBMCs were isolated by gradient centrifugation in a Ficoll-Paque PREMIUM sterile solution (GE Healthcare). Monocytes were purified using CD14 microbeads according to the manufacturer’s instructions (Invitrogen) and then were treated with 5 ng/ml recombinant human granulocyte–macrophage colony-stimulating factor (GM-CSF, switching monocytes to M1 phenotype macrophages), and cultured at 37 °C and 5% CO₂ for 5 days in the RPMI 1640 medium with 10% fetal bovine serum (FBS, Gibco).