Fixable viability dye efluor 506

Flow cytometry antibodies were purchased from eBioscience (San Diego, CA) unless otherwise specified. LSK cells that had been cultured with or without TLR agonists were analyzed by staining cells with Fixable Viability Dye eFluor^® 506, BV421-anti-CD117 (BD Pharmingen, San Jose, CA), FITC-anti-Sca-1, PE-anti-Flk2, PerCpCy5.5-anti-CD11b, PE-Cy7-anti-CD3/B220, APC-eFluor^® 780-anti-CD90.2 and Alexa Fluor 700^®-anti-CD34. Phenotyping of multiple T cell subsets was done by staining with Fixable Viability Dye eFluor^® 506, CD117-BV421 (BD Pharmingen), Alexa Fluor 700^®-anti-CD8a and Mouse Naïve/Memory T cell panel kit (BD Pharmingen), including PE-anti-CD44, PerCP-Cy^™5.5-anti-CD4, allophycocyanin-anti-CD62L and APC-Cy^™7-anti-CD3. For intracellular staining of IL-17 and IL-21, cultured cells were incubated with 50ng/ml PMA, 500ng/ml ionomycin and 1× GolgiPlug-Brefeldin A solution at 37°C for 4 hrs. Cultured cells were then harvested, surface stained with cell markers, fixed and permeabilized in fixation/permeabilization buffer (eBioscience) and stained for allophycocyanin- or FITC-conjugated anti-IL-17 or IL-21. Cells were acquired by LSRII with FACSDiva (BD, San Jose, CA) and analyzed using FlowJo (Tree Star, Ashland, OR). Flow cytometry positively stained cell gating was based on fluorescence-minus-one (FMO) samples and biological control samples.

Cryopreserved patients’ PBMCs from the same patients of different time points were thawed and cultured at the same time. PBMCs were cultured for 10 days in STEMCELL™-XF T cell expansion medium with 50IU/ml of IL-2, in the presence of 15-mer, overlapping by 11 amino acids peptide pools derived from human NY-ESO-1, MAGE-A4, or PRAME proteins (JPT, Berlin, Germany), or without peptide as a negative control. Half medium change was performed every 3–4 days. On day 10, cells were re-stimulated with the same stimulant as in culture for 6 hours, with the presence of brefeldin A (eBioscience) for the last 5 hours. Single peptide concentrations used in culture and restimulation were both 1ug/ml. Staining of CD4-BV421, CD8-APC-Cy7 and eBioscience fixable viability dye eFluor 506 was performed prior to permeabilization and fixation using BD Fixation/Permeabilization Solution Kit. IFNγ-FITC and TNF-PE-Cy7 were stained intracellularly for 30 min prior to flow cytometry analysis.