Percp cy5.5 ly 6g

For monocyte depletion studies, anti-CCR2 (clone MC-21; 25 μg) or an isotype control mAh (clone LTF-2, 25 μg) was administered to mice by intraperitoneal injection one day prior to and 1, 3, and 5 days after infection. Mice were administered 200 μg of DC2.112 or an isotype control mAb one day after subcutaneous inoculation of 10³ FFU of CHIKV AF15561 in the footpad. Joint swelling was monitored daily and at 6 dpi, mice were euthanized, extensively perfused with PBS, and tissues were collected and viral RNA quantitated. To assess the efficiency of cell depletion, peripheral blood was collected on the day of the harvest and erythrocytes were lysed with ACK Lysing Buffer (Thermo Fisher). Cells were resuspended in RPMI 1640 supplemented with 10% FBS. Single-cell suspensions were incubated with TruStain FcX (BioLegend) to block non-specific IgG binding to Fc receptors in PBS supplemented with 2% FBS for 10 min at room temperature. Cells were then stained with Fixable Viability Dye eFluor 506 (Thermo Fisher) and the following antibodies: BUV395 CD45 (BD), PE/Dazzle 594 CD11b, APC/Cy7 Ly-6C (BioLegend), and PerCP-Cy5.5 Ly-6G (BioLegend). Samples were processed on a LSR Fortessa X-20 (BD) and analyzed using FlowJo.