Phosflow perm wash buffer

Single cell suspensions were re-stimulated, surface-stained and fixed with Phosflow Lyse/Fix buffer and Phosflow Perm/Wash buffer (BD Biosciences). Cells were then stained for intracellular cytokines and transcription factors (TCF-1, Ror(γ)t and T-bet) simultaneously. Staining of FoxP3, HIF-1α and EOMES was done using a FoxP3 buffer kit (eBioscience). In some experiments, cells were first fixed with Perm/Fix buffer (BD Bioscience) followed by addition of antibodies to stain cytokines and eYFP (anti-GFP antibody), then subjected to transcription factor staining.

To measure the antigen-specific B-cell responses, we used protein labeling kits (Thermo Fisher Scientific) to conjugate Alexa Flour 647 (AF647) and Alexa Flour 548 (AF548) to extracellular domain of DENV1 (DENV1/VN/BID-V949/2007) and DENV2 (DENV2/GWL39 IND-01) E proteins (~50 kDa, CTK Biotech), respectively. One million splenocytes were incubated with DENV1/E-AF647 and DENV2/E-AF548 probes on ice for 30 min in the dark. The cells were then surface stained with conjugated anti-CD90.2, anti-F4/80, anti-CD11c, anti-CD4, anti-CD8, anti-Ly6G, anti-NK1.1, anti-IgM, anti-IgD, anti-GL7, anti-CD45R, anti-CD38 antibodies (Supplementary Table 1), and LIVE/DEAD stain FSV780 (BD Biosciences) on ice for 30 min. For the intracellular staining, cells were fixed and permeabilized with Phosflow Lyse/Fix buffer (BD Biosciences) for 10 min at 37 °C in the dark and subsequently incubated with Phosflow Perm/Wash buffer for 30 min at room temperature. Then, cells were stained with anti-Ig _(H+L) and anti-Bcl6 antibodies for 30 min at 4 °C in the dark. Cells were analyzed on an X20 flow cytometer (BD Biosciences) and data processed using FlowJo version 10.6.0 (Tree Star).