Percp anti mouse cd4

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).

The spleen single-cell suspension was prepared by 40 μM nylon cell strainer, and the red blood cells were depleted in a Tris-NH₄Cl lysis buffer (0.017 M Tris-HCl, 0.144 M NH₄Cl). The cells were incubated with FITC anti-mouse CD3, Percp anti-mouse CD4, APC anti-mouse CD25, APC anti-mouse CD8 (BioLegend, San Diego, CA, USA) at room temperature for 30 min in the dark and then washed and resuspended in 0.5 mL of PBS containing 0.5% FBS and then tested by cytometry (BD Calibur™, company, Lake Franklin, NJ, USA). The percentages of CD3⁺, CD3⁺CD4⁺, CD3⁺CD25⁺, and CD3⁺CD8⁺ expression was analyzed by the BD FACS Calibur™ CellQuest software.