Foxp3 apc clone fjk 16s

Spleens were harvested after mice were sacrificed 48 hours after CLP, and then were processed to single-cell suspensions though a 70 μm filter. Splenocytes were rinsed with 10 mL cold PBS, and 200 μL from each spleen was put into a 96-well plate for staining. Anti-CD3 (BD, clone 500A2), anti-CD4 (clone RM4-5, BioLegend), anti-CD8 (clone MCD0830, Invitrogen), anti-CD44 ( clone IM7, BioLegend), anti-CD226 (clone 10E5, BioLegend), anti-PD-1 (clone 29F.1A12, BioLegend), anti-2B4 (clone eBio244F4, Thermo Fisher Scientific), and anti-TIM-3 (clone RMT3-23, BioLegend) were used for surface staining to determine T cell phenotype. For the detection of cell apoptosis, splenocytes were stained with a FITC Annexin V apoptosis detection kit with 7-AAD (BioLegend). Tregs were identified via intracellular staining for Foxp3-APC (clone FJK-16S, eBioscience). Splenocytes were surface stained for anti-CD62L (MEL-14), anti-CD69 (H1.2F3), and then permeabilized using a Foxp3 kit (BD Biosciences) and stained with anti-Foxp3, anti-CTLA-4 (UC10-489), and anti-Helios (22F6, all Abs from BioLegend). AccuCheck Counting Beads (Thermo Fisher Scientific) were added after staining to calculate the absolute number of cells per spleen.

Cell suspensions from iliac lymph nodes, blood and tumors were first incubated with 1µg/10⁶ cells of Fc block treatment (BD Pharmigen) and then stained for surface markers in FACS buffer (PBS + 1% FBS). For Treg staining on cells isolated from iliac lymph nodes: Live/Dead (Fixable Near-IR Dead Cell stain kit, Life Technologies); CD3 FITC, clone 145-2C11 (Biolegend); CD4 Pacific Blue, clone RM4-5 (Biolegend); Foxp3 APC, clone FJK-16S (eBioscience). For Treg staining on cells isolated from tumors: Live/Dead (Fixable Near-IR Dead Cell stain kit, Life Technologies); CD45 PerCP-Cyanine 5.5 (eBioscience); CD4 Pacific Blue, (Biolegend); CD8 Brilliant Violet 510, clone 53-6.7 (Biolegend); Foxp3 APC, (eBioscience). For effector memory staining on cells isolated from blood: Live/Dead (Fixable Near-IR); CD8 PerCP, (Biolegend); CD44 APC (Biolegend); CD62L FITC, clone MEL-14 (BD Pharmigen).