Cd4 percep cyanine 5.5 rm4 5

Flow cytometry analyses were performed as described previously (Jang et al., 2019 (link); Kim et al., 2019 (link)). Briefly, MLN tissues were smashed and filtered using a cell strainer (100 μm pore size (SPL Life Sciences Co., Ltd., Pocheon-si, Gyeonggi-do, Republic of Korea) and isolated T cells were subjected to a FC gamma receptor blocking and surface staining for 30 min at 4°C. To identify the live cells, T cells were stained with Fixable Viability Stain 510 (FVS510; BD Biosciences, San Jose, CA, USA) following the manufacturer's instructions. Moreover, T cell surface were stained with CD3+ fluorescein isothiocyanate (145-2C11; BD Biosciences), CD4+ Percep-Cyanine 5.5 (RM4-5; BD Biosciences) and CD25+ phycoerythrin (PC61; BD Biosciences) following the manufacturer's instructions. T cells were permeabilized with fixation/permeabilization buffer (eBioscience, San Diego, CA, USA) for intracellular staining and were stained with Foxp3+ Alexa Flour 647 (MF23; BD Biosciences) following the manufacturer's instructions. IgG isotypes (BD Biosciences) were used as a control. The CD4+CD25+Foxp3+ T cell population was counted using a BD FACSVerse™ Flow Cytometer (BD Biosciences).

Flow cytometric analyses were carried out as described previously.^{46 (link)} Briefly, isolated MLN cells were stained with Fixable Viability Stain 510 (FVS510; BD bioscience, Franklin Lakes, NJ, USA) for live cells, as well as CD3+ fluorescein isothiocyanate (145–2 C11; BD bioscience), CD4+ Percep-Cyanine5.5 (RM4–5; BD bioscience) and CD25+ phycoerythrin (PC61; BD Bioscience) for cell surface staining after blocking with FcγR and permeabilization with fixation/permeabilization buffer (Ebioscience, San Diego, CA, USA) and were also treated with Foxp3+ Alexa Fluor 647 (MF23; BD Bioscience) for intracellular staining. IgG isotypes were used as a control in all flow cytometry experiments. The CD4+ CD25+ Foxp3+ Treg population was analyzed using a BD FACSVerse™ Flow Cytometer (BD Bioscience).