Cyan adp dako

Cell proliferation and viability was assessed using a Countess Automated Cell Counter (Life Technologies) and Tripan Blue exclusion assay according to manufacturer instruction. For cell cycle analysis, 2 × 10⁵ cells were resuspended in PBS 50% FCS, fixed in 70% ethanol for 24 hours, incubated with 50 μg/mL propidium iodide (Sigma-Aldrich) and 50 units/mL DNase free RNase A (Sigma-Aldrich), and analyzed after 3 hours (10, 000 events) using an Epics XL Cytometer (Beckman Coulter). Data were analyzed with the FCS Express 4 Flow. A minimum of 10,000 events was collected for each sample with flow cytometer (CyAN ADP DAKO) by using Summit 4.3 software for data acquisition and analysis.

For cell cycle analysis, 2 × 10⁵ cells were resuspended in 50% FCS, fixed in 70% ethanol for 24 hours, incubated with 50 Ag/mL propidium iodide (Sigma-Aldrich) and 50 units/mL DNase free RNase A (Sigma-Aldrich), and analyzed after 3 hours (10,000 events) using an Epics XL Cytometer (Beckman Coulter). Differentiation was assessed by NBT dye reduction assay and direct immunofluorescence staining of cells using an Allophycocyanin (APC) anti-human CD11b (Becton Dickinson), APC anti-human PerCP-Cy5.5 anti-human CD14 (Becton Dickinson) and PE-IgG1 isotype control (eBiosciences) as previously described (Salvatori et al., 2012). A minimum of 10,000 events was collected for each sample with flow cytometer (CyAN ADP DAKO) by using Summit 4.3 software for data acquisition and analysis.

Data for growth curves and cell viability were obtained with the Countess II FL Automated Cell Counter (Thermo Fisher Scientific, Waltham, MA, USA), excluding the trypan blue positive cells. Cell cycle was analyzed by flow cytometry (CyAN ADP DAKO, Beckman Coulter, Brea, CA, USA) as previously described [19 (link)].