Flow Cytometric Analysis of Immune Cells in CHIKV-Infected Mice

Spleen, popliteal lymph node (pLN) and joint footpad of CHIKV-infected mice were harvested at 6 dpi and 7 dpi. Isolation of splenocytes, pLN and joint footpad cells were processed as described4 (link)21 (link)26 (link). Isolated cells from spleen, joint footpad and pLN were first stained with Live/dead Fixable Aqua Dead Cell Stain Kit (1:400) (Life Technologies) for 20 min. Cell were then washed and resuspended in staining buffer (2% fetal bovine serum in PBS). Staining was performed using rat anti-CD45 (30-F11, BD Pharmingen), rat anti-CD4 (RM4–5, BD Pharmingen), rat anti-CD3 (17A2, Biolegend), rat anti-CD25 (PC61.5, eBioscience), hamster anti-CTLA-4 (UC10-4B9, eBioscience) and rat anti-CD44 (IM7, eBioscience) antibodies for 15 min. Stained cells were then washed with PBS and fixed using IC Fixation Buffer (eBioscience). Intracellular staining of rat anti-Foxp3 (FJK-16s, eBioscience) was done according to manufacturer’s instructions. Data acquisition and analyzes were done as described above.