Flow cytometric analysis of peripheral blood lymphocytes
LYSED, BLOOD, LYSING, APC
To evaluate the relative proportions of lymphocyte populations in peripheral blood, the following panel of fluorochrome conjugated antibodies was used: anti CD45 BUV 395, anti CD3 APC R700, anti CD19 APC, anti CD16 PE-Cy7, anti CD56 BB515, anti CD4 BUV 7373, anti CD8 BV 785, anti CD25 BV 421, anti CD127 BB700, anti CD45RA BV605, anti CD197 BV711, anti FoxP3 PE, anti RORγT BV650 (all from BD Biosciences, Milan, Italy). Blood samples were stained with anti membrane associated molecules and, after incubation, erythrocytes were lysed using FACS lysing solution, according to manufacturer’s instruction. To analyze the expression of transcription factors (FoxP3 and RORγT), samples were fixed and permeabilized using Transcription Facytors Buffer Set (BD Bioscience), according to standard protocol. Samples were acquired and analyzed on a LSR Fortessa X-20 (Becton Dickinson), using FACS DIVA v8.0.2 software.