Facsaria iiu analyzer

Human colon cancer cells derived from monolayer cultures and sphere-derived cells on day 7 after primary culture were adjusted to a final concentration of 10⁶ cells/ml. Cell suspensions were incubated for 10 min with blocking solution and with appropriate antibodies in the dark at 4°C for 30 min. Cells were then washed with PBS and analyzed by flow cytometry (BD Accuri™ C6, BD Biosciences). We repeated each characterization three times to validate the results observed. We followed the protocol of Miranda-Lorenzo et al. (2014) (link) to acquire autofluorescent cells from cultured cells: 30 µM riboflavin (Sigma-Aldrich) was added to cultured cells. For FACS acquisition cells were incubated overnight at 37°C, centrifuged at 300 g for 5 min and cell pellets re-suspended in PBS. Autofluorescent cells were excited with 488–561 nm laser and selected as the intersection with filters 496/578. Propidium iodide (Sigma-Aldrich) was used for exclusion of dead cells. Cell sorting was performed in a FACSAria IIu analyzer (BD Biosciences) by using the PC FACSDiva software program (BD Biosciences).

Circulating EPC leves were measured by flow cytometry according to methods described elsewhere33 (link) in blood samples obtained at admission, 72 hours and at day 7. Blood samples were processed within 1–2 hours after collection by a single researcher who had no knowledge of the patients’ clinical or radiological results. In brief, circulating EPCs were analyzed for the expression of specific surface antigens using direct flow cytometry (BD FACSAria IIu, BD, Franklin Lakes, NJ, USA). Cells were labelled with FITC-conjugated anti-CD34 (BD, Franklin Lakes, NJ, USA), PE-conjugated anti-KDR (R&D Systems, Minneapolis, MN, USA) and APC-conjugated anti-CD133 (clone AC133 from Miltenyi Biotec, Bergisch Gladbach, Germany) monoclonal antibodies. We considered EPCs as triple CD133⁺/CD34⁺/KDR⁺ staining cells in the mononuclear cell fraction. In all analyses, 5 × 10⁵ events were acquired, scored using a FACSAria IIu analyzer (BD, Franklin Lakes, NJ, USA), and processed using the PC FACSDiva software program (BD, Franklin Lakes, NJ, USA). Cell count was always expressed per 10⁶ events.