Anti foxp3 pe

Cells were stained with PE-Cy7 anti-F4/80, PE-Cy7 anti-CD11c, PerCP anti-CD3, PE anti-PD-L1, APC anti-CD86, FITC-CD206, PE anti-Foxp3, APC anti-CD25, and APC anti-IFNγ (BioLegend, San Diego, CA). Labile cell iron was measured using 10 μM calcein acetoxymethyl (calcein_AM, Thermo Fisher Scientific, MA). Lipid peroxidation was assessed by C11-BODIPY 581/591 (Thermo Fisher Scientific, MA), and the amount of ROS was measured by a DCFDA cellular ROS detection assay kit (Abcam, Cambridge, UK). CD4⁺IFNγ⁺ cells were detected by an intracellular staining kit (Fixation/Permeabilization Solution Kit, Franklin Lakes, NJ), and CD4⁺CD25⁺Foxp3⁺ cells were measured using a Foxp3/transcription factor staining buffer set (eBioscience). All data were analyzed using FACS LSR Fortessa cytometry with BD CELL Quest Pro software.

Flow cytometry was performed using FACS LSR2 (BD Biosciences, CA, USA). Data were analyzed using FlowJo software (FlowJo, LLC, OR, USA). Fluorescence-conjugated monoclonal antibodies were purchased from the following sources: Human LAG-3 Alexa Fluor® 488-conjugated Antibody (R&D system, MN, USA); PE/Cy7 anti-CD3, FITC anti-CD4, PE anti-CD8, FITC anti-CD45RO, FITC anti-HLA-DR, APC anti-CD25, APC/Cy7 anti-CD69, APC anti-human CD279 (PD-1), APC/Cy7 anti-human CD366 (Tim-3), and PE anti-FoxP3 (BioLegend, CA, USA). Furthermore, cells were stained with PE anti-human CD274 (B7-H1, PD-L1, BioLegend, CA, USA) to identify proportion of PD-L1 positive tumor cells in body fluid.
For Treg cell staining, cells were stained with various antibodies except FoxP3 antibody for which cells were fixed and permeabilized with eBioscience™ FoxP3 Fixation/Permeabilization solution (Thermo Fisher Scientific, MA, USA). FoxP3 antibodies were administered after permeabilization for intracellular staining of Tregs. FACS analyses were performed for cells isolated from malignant fluid and peripheral blood. First, levels of CD4+ and CD25+ T cells in cells isolated from these two sources (malignant fluid and peripheral blood) were measured. Next, we quantified the percentage of cells that were positive for FoxP3 in the CD4 + CD25+ T cell population.

Dextran sulfate sodium (DSS, MW 36000-50000) was obtained from MP Biomedicals (CA, USA). The myeloperoxidase (MPO) assay kit was obtained from Jiancheng Bioengineering Institute (Nanjing, China). Zonula occludens protein 1 (ZO-1) and Occludin antibodies were obtained from Affinity (Liyang, China). FITC anti-CD4, PE anti-Foxp3, and PE anti-IL-17A antibodies were obtained from Biolegend (CA, USA). CD3e antibody, APC anti-CD25 antibody, Foxp3/transcription factor staining buffer set and IC fixation buffer were obtained from eBioscience™ (CA, USA). CD16/CD32 antibody was obtained from BD Biosciences (CA, USA). STAT3 and phospho-STAT3 (p-STAT3) antibodies were obtained from Cell Signaling Technology (MA, USA). The QuantiCyto^® Mouse TNF-α enzyme-linked immunosorbent assay (ELISA) kit was obtained from NeoBioscience (Shenzhen, China).

5. -AIQ was obtained from Matrix Scientific (Columbia, SC, USA. Roswell Park Memorial Institute). RPMI 1640 medium was purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies to FOXP3 (#SC-130666), Helios (#SC-390357), GATA3 (#SC-268), and IL-17A (#SC-374218) were purchased from Santa Cruz Biotech, (Dallas, TX, USA). GolgiStop was purchased from BD Biosciences (San Diego, CA, USA). Conjugated phycoerythrin (PE), fluoro-isothiocyanate (FITC), PE/Dazzle 594, allophycocyanin (APC). APC anti-CD4 (#100412), FITC anti-CD4 (#100510), APC anti-CXCR6 (#151106), FITC anti-CXCR6 (#151107), PE anti-FOXP3 (#126404), PE anti-Helios (#137206), APC anti-GATA3 (#653806), PE anti-IL-17A (#506903), PE anti-IL-9 (#514104), and APC anti-IL-10 (#505010) monoclonal antibodies, red blood cell lysis buffer, fixation buffer, and intracellular staining permeabilization buffer were all obtained from BioLegend (San Diego, CA, USA). TRIzol reagent was purchased from Invitrogen (Carlsbad, CA, USA). SYBR Green and High-Capacity cDNA reverse transcription kit were purchased from Applied Biosystems (Foster City, CA, USA). Primers were synthesized by GenScript (Piscataway, NJ, USA). Nitrocellulose membranes were obtained from Bio-Rad Laboratories (Hercules, CA, USA). Western blot chemiluminescence kit was purchased from Millipore (Billerica, MA, USA).