Foxp3 kit

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.

20 μg/mL PMA and 1 μg/mL of ionomycin were used to stimulate splenocytes in the presence of GolgiStop (BD Biosciences). The cells were then fixed and permeabilized using a Foxp3 kit (BD Biosciences). Intracellular cytokine staining was then performed using anti- IL-2 (BioLegend, clone JES6-5H4), anti-IL-10 (BioLegend, clone JES5-16E3), anti-IFNγ (BD, clone XMG1.2), anti-TNF (BioLegend, clone MP6-XT22), anti-IL-6 (Invitrogen, clone MP5-20F3), and anti-IL-1β (Invitrogen, clone NJTEN3).

24hrs after CLP, mice were euthanized, and their spleens were harvested for processing into single-cell suspensions using a 70 μm filter. Splenocytes were rinsed and resuspended in 10 mL of PBS with 200 μL of each sample placed into a 96-well plate for staining. Anti-CD3 (BioLegend, clone 17A2), anti-CD8 (BD, clone 53-6.7), anti-CD4 (Invitrogen, clone RM4-5), anti-CD44 (BioLegend, clone IM7), anti-TIGIT (BD, clone 1G9), anti-PD-1 (BioLegend, clone 29F.1A12), anti-CD25 (BioLegend, clone PC61), anti-CD69 (BioLegend, clone H1.2F3), and anti-LAP (Thermo Fisher Scientific, clone TW7-16B4) were used for cell surface immunophenotyping. Anti-Foxp3 (Invitrogen, clone FJK-16s) was used for identification of regulatory T cells after splenocytes were fixed and permeabilized (Foxp3 kit, BD Biosciences). CountBright™ Absolute Counting Beads (Thermo Fisher Scientific) were added to samples after staining to calculate the absolute numbers of cells per spleen. Samples were run on a Fortessa flow cytometer (Becton Dickinson) and data was analyzed using FlowJo software (ver. 10.8.1).