Biocoll 1077

Blood centrifugation on a density gradient (Biocoll 1077, Biochrom, Cambridge, UK) was used to isolate PBMCs. The relative intracellular cholesterol (IC) level in PBMCs was determined in all participants before and after 6 weeks of SLO supplementation. IC level was evaluated using the cholesterol assay kit, Cholesterol Chod-PAP (BIOLABO S.A., Maizy, France) per the manufacturer’s recommendations. These results were then normalized according to the protein concentration of each PBMC sample that was determined by Bradford assay (Bio-Rad, Hercules, CA, USA), as previously described [31 (link)].

Bone marrow mononuclear cells were separated by density-gradient centrifugation (Biocoll 1.077 g/mL, Biochrom GmbH, Berlin, Germany) and seeded in growth medium (DMEM low glucose [Sigma-Aldrich, St. Louis, MO, USA], 20% foetal bovine serum [FBS Superior, Biochrom GmbH], and 1% penicillin/streptomycin/L-glutamine [Sigma-Aldrich]) in a humidified atmosphere at 37°C and 5% CO₂. After one week nonadherent cells were removed, and growth medium was changed every 3-4 days. Adherent cells were passaged weekly and seeded at 5000 cells/cm². Experiments were carried out using MSCs derived from passage 3. Before that, the MSC character of the cultured cells was determined and possible contamination with haematopoietic cells excluded by flow cytometry (≥95% expression of CD73, CD90, and CD105, while lacking CD34 and CD45).

For CTC isolation, blood samples were layered over 4 ml polysucrose solution (Biocoll 1077, Biochrom, Berlin, Germany) and centrifuged for 20 min at 2500×g. Peripheral blood mononuclear cells (PBMCs) and CTCs (the buffy coat) were collected and washed with phosphate-buffered saline (PBS, P3813, Sigma-Aldrich, Germany). Cell pellets were resuspended for 10 min in erythrocyte lysis buffer [154 mM NH₄Cl (31,107, Sigma-Aldrich, Germany), 10 mM KHCO₃ (4854, Merck, Germany) and 0.1 mM EDTA]. Cells were then collected by centrifugation, washed in PBS, and incubated with mouse monoclonal anti-human CD45 antibody-conjugated magnetic beads (39-CD45–250, Gentaur, Belgium) for 30 min at 4 °C. Anti CD45 bead-bound cells were collected in a magnetic field and saved for use as non-cancer control PBMCs in qRT-PCR gene expression analyses. Remaining cells were incubated with mouse monoclonal Anti-Pan cytokeratin-conjugated microbeads (PCK/CK4, CK5, CK6, CK8, CK10, CK13 and CK18) (MA1081-M, Gentaur, Belgium) for 30 min at 4 °C and PCK bead-bound cells (CTCs) collected in a magnetic field and washed in PBS. Isolated viable CTCs (IV-CTCs) were counted, and samples containing ≥5 viable CTCs per ml of blood were cultured for 6 days in order to obtain sufficient cell numbers for gene expression and chemosensitivity assays.