α cd4 bv711

To assess the therapeutic value and safety of (R)-DI-87, cohorts of female C57BL/6 mice were treated with (R)-DI-87 (75 mg/kg) or vehicle (40% Captisol) via oral gavage in 12-hour intervals over the course of 16 days. On day 16, 100 μl peripheral blood was collected in lithium heparin-coated tubes via retro-orbital bleeding using heparin-coated capillary tubes. Subsequently, blood samples were incubated with 5 ml of ACK lysis buffer at room temperature for 5 min, quenched with 5 ml of FACS buffer (5% FBS in PBS), and centrifuged at 4°C (4 min). This process was repeated and cells were subsequently stained with the following fluorochrome-conjugated anti-mouse antibodies diluted 1:100 in 100 μl of FACS buffer for 20 min at 4°C: α-B220-PerCP/Cy5.5 (103236, BioLegend), α-CD4-BV711 (100550, BioLegend), α-CD8α-PE (100708, BioLegend), α-CD11c-PE/Dazzle594 (117347, BioLegend), α-CD11b-FITC (101206, BioLegend), α-GR1-APC (108412, BioLegend); α-CD16/32 (FC block; 101319; BioLegend). Following incubation, cells were centrifuged and washed twice using FACS buffer. Cells were resuspended in FACS buffer and analyzed using a BD LSRII flow cytometer and the FlowJo software package. During the (R)-DI-87 or vehicle treatment procedure, mice were regularly monitored and weighed to assess the overall health status.