Apc cy7 anti mouse cd25

Twenty four weeks post final DNA immunization, the splenocytes were isolated from the immunized mice (n = 6 per group). CD4⁺ memory cells were stained with PerCP-Cy5.5-anti-mouse CD4 (Biolegend), FITC-anti-mouse CD44 (Biolegend), PE-anti-mouse CD62L (Biolegend), and APC-Cy7-anti-mouse CD25 (Biolegend). CD8⁺ memory cells were stained with PerCP-anti-mouse CD8 (Biolegend), PE-anti-mouse CD62L (Biolegend), and Pacific blue-anti-mouse CD45R (Biolegend) antibodies. The stained cells were fixed with Cytofix/Cytoperm Buffer™ (Becton Dickson) and then analyzed with a FACS Canto II flow cytometer with FACSDiva software.

To differentiate CD4⁺ T cells into a T regulatory cell phenotype, splenocytes from SMARTA triple-reporter mice were processed and the red blood cell lysed using an ACK (ammonium-chloride-potassium) lysis buffer. The cells were then activated with 1 μg/mL GP_61–80 peptide (GenScript, Piscataway, NJ) and 5 ng/mL TGF-β1 (Shenandoah Biotechnology, Inc., Warwick, PA). After one day of initial skewing, 100 μg/mL IL-2 along with ATO or Mito-ATO analogs of varying concentrations were added to the culture. Cells were cultured for six days and split once cells reached confluency; cells were replenished with IL-2 and compound accordingly. After six days in culture, cells were stained to assess the viability and phenotypic analysis via flow cytometry. LIVE/DEAD fixable violet or aqua dead cell stain (Invitrogen, Carlsbad, CA) was used to assess cell viability. The following antibodies were used for flow cytometry staining: PercP anti-mouse CD4 (clone: GK1.5; BioLegend, San Diego, CA), APC/Cy7 anti-mouse CD25 (clone: PC61; BioLegend), and PE anti-mouse FOXP3 (clone: FJK-16S; eBioscience, San Diego, CA). Flow cytometry data were acquired using a BD LSRII (BD Biosciences, CA) flow cytometer and analysed using FlowJo (Treestar, Inc., Ashland, OR).