Cytofix buffer

For intracellular STAT3 and phosphorylated STAT3 (pSTAT3) level analysis, freshly isolated PBMCs were suspended in complete RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin and streptomycin (all from Sigma-Aldrich Inc., St Louis, MO, USA). 1x10⁶ PBMCs in 1 ml were either left unstimulated or were stimulated with 10 µg/ml phytohaemagglutinin (PHA, Sigma-Aldrich Inc.) for 72 hours. PMBCs were then washed and fixed with BD Cytofix™ buffer (BD Biosciences, Franklin Lakes, NJ, USA) for 20 minutes. Following the washing step, fixed cells were permeabilized by using BD Phosflow™ Perm Buffer III (BD Biosciences) for 10 minutes. Unstimulated and PHA stimulated cells were then stained with allophycocyanin (APC) conjugated anti-STAT3 (M59-50) monoclonal antibody which recognizes STAT3 regardless of phosphorylation status. For the detection of phosphorylated STAT3, phycoerythrin (PE) conjugated anti-STAT3 (pS727) monoclonal antibody which recognizes the S727-phosphorylated form of STAT3 isoform 1 was used. All samples were also stained with isotypic controls. STAT3 and pSTAT3 levels of PBMCs were analysed with FACSCanto flow cytometry using Diva software (BD Biosciences). Results were expressed as median (range) of pSTAT3/STAT3-expressing mononuclear cells in PB and non-parametric Mann-Whitney-U test was used for comparisons.

The antibodies for flow cytometry are summarized in the Key Resources Table. Cells were stained with fluorochrome-conjugated antibodies against surface antigens in DPBS (Thermo Fisher Scientific) containing 2% FBS (Thermo Fisher Scientific) prior to analysis, cell sorting or intracellular staining. To stain intracellular Igμ, Igk or VPREB, cells were fixed and permeabilized with a fixation/permeabilization solution (BD Biosciences) before being stained for intracellular proteins. To stain intracellular FOXO1, cyclin D3 or IKZF3, cells were fixed and permeabilized with the True-Nuclear transcription factor buffer set (BioLegend). For FOXO1 staining, after fixation and permeabilization, cells were stained with the Rabbit anti-FOXO1 antibody and washed three times, followed by being stained with a FITC-labeled anti-Rabbit IgG. For intracellular staining of p-STAT5, p-AKT or p-ERK, cells were fixed with BD Cytofix buffer and permeabilized with BD Phosflow Perm Buffer III as recommended by the manufacturer (BD Biosciences). Labeled cells were analyzed on a BD LSR II or a BD LSRFortessa, or sorted on a BD FACSAria, using Diva software (BD Biosciences). Flow cyto-metric data were analyzed using FlowJo software (BD Biosciences).