Cyan adp 9 colors cytometer

Spleen were mechanically dissociated to yield a single-cell suspension and were treated with ammonium chloride–potassium buffer for red blood cell lysis. After isolation, cell suspensions were passed through 40μm cell strainers (BD Pharmingen) and cell populations were characterized by flow cytometry. The cells were then blocked with Fc-block (BD Biosciences) and stained with anti-mouse antibodies according to the manufacturer’s instructions. Stained samples were analyzed on Cyan ADP 9 colors cytometer (Beckman Coulter) and analyses were performed with FlowJo software version 10. For intracellular staining of produced cytokines, cells were first stimulated with 1 μL PMA/Ionomycin Mixture (250×) and 1 μL BFA/Monensin Mixture (250×). With mononuclear cells only as control. Mix wells were incubated in CO2 incubator at 37°C for 4–6 hours and vortexed every 1–2 hours during incubation, according to the manufacturer’s instructions. Data were acquired using LSRII (BD Bioscience) and analyzed by FlowJo software v9.6.2. (Tree Star, Inc., Ashland, OR).