*4.8. STATs Flow Cytometry*

The spontaneous and stimulated CD4+T-lymphocyte subset expression of STAT-1, STAT-3 and STAT-6 was assessed by in vitro phosphoprotein intracytoplasmic staining. To evaluate STAT-1, STAT-3 and STAT-6 phosphorylation, PBMCs were stimulated for 15 with 40.000 U/mL of IFNα for STAT-1, 0.1μg/mL of IL-6 for STAT-3 and 0.1 μg/mL of IL-4 for STAT-6. Additionally, the total STAT-1, STAT-3 and STAT-6 protein was analyzed in CD4+T-lymphocyte subsets.

Surface and intracellular lymphocyte staining. For intracytoplasmic STAT staining, PBMCs were fixed, permed (BDPhosphoflow, Becton-Dickinson, BD, San Jose, CA, USA) and stained with the subsequent surface-labeled monoclonal-antibodies CD4-FITC, CD27-PE, CD3-Percp (Becton-Dickinson, BD, San Jose, CA, USA) CD45RA-Alexa405 (eBioscience, San Diego, CA, USA) and with the STAT1-FITC, STAT3-Pacificblue or STAT6-PE (Becton-Dickinson, BD, San Jose, CA, USA) intracellular-labeled monoclonal-antibodies against phosphorylated or total STAT protein.

The quality control of the flow cytometer was performed daily according to the manufacturer's instructions (Becton-Dickinson, BD, San Jose, CA, USA). The staining protocol and quality and analysis controls were performed by 'fluorescence minus one control' as described by Roederer et al. [49], and the flow cytometry results were presented following the guidelines of the International Society of Advancement of Cytometry (ISAC) [50].
