Splenic Leukocyte Isolation and Tetramer Staining

Spleens were removed aseptically and transferred to Hanks Balanced Salt Solution (HBSS). Single-cell suspensions were obtained by pressing the spleens through a 70-µm nylon cell strainer, followed by centrifugation and two washes in HBSS. Approximately 2 × 10⁶ splenocytes were transferred to U-bottom 96-well microtiter plates and were incubated for 20 min (4°C in the dark) with PE-conjugated H-2D^b tetramers for ZIKV E_294–302 (25 (link)) and subsequently stained for an additional 20 min (4°C in the dark) for relevant cell-surface markers. Cells to be stained for intracellular granzyme B were then permeabilized using the BD Biosciences FoxP3 staining protocol. Next, the cells were centrifuged, washed, fixed in 1% PFA, and finally resuspended in PBS and stored at 4°C until flow cytometric analysis. Cell samples were analyzed using FACS LSRFortessa cytometer (BD Biosciences), and the data were analyzed using FlowJo software version 10 (Tree Star).