Fitc labeled anti cd25

For cell phenotypic and intracellular cytokines study and transcription factor analysis, FITC-labeled anti-CD3, APC-Cy7-labelled anti-CD4, PE labeled anti-CD19, FITC-labeled anti-CD25, PE-Cy7-labelled anti-CD69, PE-Cy7-labelled anti-IFN-γ, APC-labeled anti-IL-4, PE-labeled anti-IL-9 were obtained from BD Biosciences (San Jose, CA, US). For Western blotting, mouse anti-PU.1 mAb was purchased from Cell Signaling Technology (Danvers, MA, US). Mouse anti-β-actin and anti-mouse IgG-HRP were bought from Santa Cruz Biotechnology (Santa Cruz, CA, US). Human ELSIA kits for cytokine IFN-γ, IL-4, IL-9, IgG and IgE were purchased from BD Biosciences. ELISpot kits for cytokine IFN-γ, IL-4 were purchased from BD Biosciences. IL-9 ELISpot Kit was produced by R&D Systems (Minneapolis, MN, US). Purified anti-CD3, anti-CD28 and anti-CD40 monoclonal antibodies (mAbs) were from the production of BD Biosciences. Recombination IL-9 and IL-4 were from Peprotech (Rocky Hill, NJ, US). House dust mite extract was purchased from GREER Company (Lenoir, NC, US).PMA, ionomycin and Brefeldin A were produced by Sigma-Aldrich (St. Louis, US).

After 7 days of expansion, T cells were harvested and immunophenotyping was performed. T cells were incubated with the following monoclonal antibodies: fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, PE-cyanine 7 (PE-Cy7)-, and V450-labeled anti-CD3; allophycocyanin (APC)-, and Alexa Fluor 700-labeled anti-CD4; Peridinin-chloropyll-protein complex (PerCP)-labeled anti-7-Aminoactinomycin D (7-AAD); FITC-labeled anti-CD28; FITC-labeled anti-CD69; FITC-labeled anti-CD94; FITC-labeled anti-CD56; FITC-labeled anti-TCRαβ; FITC-labeled anti-CD95; APC-labeled anti-CD45RO; PE-Cy7-labeled anti-CCR7; FITC-labeled anti-CD25; APC-labeled Annexin V (all from BD Bioscience, Franklin Lakes, NJ, USA); FITC-labeled anti-TCRγδ (Becman Coulter, Fullerton, CA, USA); APC-Alexa Fluor 700-labeled anti-CD127 (Beckman Coulter, Fullerton, CA, USA); Pacific Orange-labeled anti-CD8 (Invitrogen); PE-labeled anti-CD39 (BD Bioscience, Franklin lakes, NJ, USA) for 15 min and washed before flow cytometry (FACS Aria flow cytometer, BD Biosciences) and analysis (FlowJo software, Tree Star, Inc., Ashland, OR, USA). T cell death was assessed by flow cytometry staining for 7-AAD and Annexin V after 7 days culture with beads and cytokines followed by a brief (6 h) period of stimulation with peptide mix spanning the cytomegalovirus (CMV) protein pp65.