Analyzing Th17 and Th1 Cells in B-ALL

The frequencies of Th17 cells and Th1 cells were analyzed as previously described [7 (link)]. Briefly, peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMCs) from B-ALL patients and healthy donors were stimulated with 40 ng/ml phorbol 12-myristate13-acetate (PMA) and 1 μg/ml ionomycin for 5 h in the presence of 1 μg/ml brefeldin A (BFA; all from Sigma-Aldrich, St.louis, MO, USA). The cells were subsequently surface stained with a combination of PerCP-conjugated anti-CD3 and FITC-conjugated anti-CD8, fixed and permeabilized and intracellularly stained with PE-conjugated anti-IL-17A and APC-conjugated anti-IFN-γ (all from BD Biosciences, San Jose, CA, USA). Stained cells were acquired and analyzed using CellQuest software on a FACSCalibur instrument (BD Biosciences).