Rat anti cd25 pc61

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.

Fifteen microliters of blood were collected from the tail vein. Briefly, blood was lysed using 1X Mouse Lyse Buffer (R&D Systems) and washed twice with PBS. Cells were stained with rat anti-CD4 (clone RM4–5, BD Biosciences), rat anti-CD25 (PC61.5, eBioscience), rat anti-CD44 (IM7, eBioscience) antibodies. Intracellular staining of Foxp3 was done using Foxp3 staining buffer set (eBioscience) and rat anti-Foxp3 (FJK-16s, eBioscience) following manufacturer’s instructions. Data was acquired using BD LSRFortessa™ cell analyzer. Dead cells and duplets were excluded in all analyzes using forward and side scatter gating. Results were analyzed with FlowJo version X software (Tree Star, Inc).