Anti cd4 apc cy7 rpa t4

The samples were ex-vivo aspirated from the resected thyroid glands in the operation room and immediately mixed in RPMI media containing 10% FCS at 4°C. The red blood cells were lysed by a brief hypotonic shock. For all flow cytometry experiments, cells were stained with fluorochrome-conjugated antibodies against human CD3, CD4, CD8, CD14, CD19, CD25, CD45, CD56, and iNKT (anti-CD3-allophycocyanin/HIT3a, anti-CD4-APC-cy7/RPA-T4, anti-CD8-PE-cy5/HIT8a, anti-CD14-PE/M5E2, anti-CD19-PE-cy5/HIB19, anti-CD25-PE/M-A251, anti-CD45-FITC/HI30, anti-CD56-PE-cy7/B159, anti-iNKT FITC/6B11, TCRαβ-FITC/T10B9.1A-31 and TCRγδ-PE/B1; BD Biosciences) or isotype controls in serum-containing media. Freshly isolated single cells were incubated with antibodies for 20 minutes on ice for surface staining, washed and fixed in 1% paraformaldehyde. A subset of cells was permeabilized with cytofix/cytosperm fixation and permeabilization solution (BD Biosciences) and stained with fluorochrome-conjugated antibodies against human IL17, IFNg and Foxp3 (anti-IL17-PE/N49-653, anti-IFNg-PE/25723.11, anti- Foxp3-Alexa Fluor 488/259D/C7; BD Biosciences). Cells were also stained with Hoechst 33342 (10μg/ml for 2 hr, Hoechst fluorescence, 350 nm excitation/ 450 nm emission, linear scale) used to gate live cells containing 2n-4n cellular DNA.