Foxp3 staining protocol

Approximately 2-5x10⁶ cells from the brain samples or 2x10⁶ splenocytes were transferred to wells of U-bottomed 96-well microtiter plates. For tetramer staining the cells were incubated for 20 min (RT, in the dark) with 50 μl FACS medium (PBS, 1% BSA, 0.1% N_aN₃) containing 10 µL relevant fluorochome labeled tetramers; these were kindly provided by S Buus and A Stryhn (this institute). To prevent unspecific binding cells were blocked with α-CD16/32 and subsequently incubated for 20 min (4°C, in the dark) with 50 µL brilliant violet staining buffer (Biolegend) containing conjugated antibodies (1:100) for the relevant cell-surface markers. Cells to be stained for intracellular granzyme B, T-bet or Eomes expression were permeabilized and stained according to the FoxP3 staining protocol from BD Biosciences. When analyzing the EdU incorporation cells were permeabilized and stained according to the Click-It™ Plus EdU Alexa Flour™ 647 Flow Cytometry Assay Kit from Invitrogen by Thermo Fisher Scientific. Cells were subsequently washed twice in wash media (PBS with 0.1% N_aN₃), resuspended in PBS and stored at 4°C until analysis. The general gating strategy regarding CNS derived cells is depicted in Supplementary Figure 1.

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).

Following preparation of single cell suspensions, approximately 2–5 × 10⁶ cells from the brain samples or 2 × 10⁶ spleenocytes were transferred to wells of U-buttomed 96-well microtiter plates. For surface staining, the cells were incubated for 20 min (4°C, in the dark) with 50 μl FACS medium (PBS, 1% BSA, 0.1% N_aN₃) containing 10 μL relevant fluorochome labeled tetramers (30 (link), 31 (link)); these were kindly provided by S Buus and A Stryhn (this institute) (34 (link)). Additionally the cells were incubated for 20 min (4°C, in the dark) with 50 μL brilliant violet staining buffer (Biolegend) containing conjugated antibodies (1:100) for the relevant cell-surface markers and unconjugated CD16/32 to prevent unspecific binding. For many of the phenotypic analyses, the pattern for two different antigen specific subsets was evaluated; only subtle differences were noted. Cells to be stained for intracellular Granzyme B, Bcl-2 or Ki-67 expression were permeabilized and stained according to the FoxP3 staining protocol from BD Biosciences. After incubation, the cells were washed twice in wash media (PBS with 0.1% N_aN₃), resuspended in PBS and stored at 4°C until flow cytometric analysis later the same day.