Anti foxp3 alexa fluor 488 clone fjk 16s

Mediastinal lymph nodes were removed and processed similarly to lung as described in Section “Cytokine Levels in Lung Tissues.” The cells were collected, and the remaining erythrocytes of the left lung were lysed (lysis buffer, eBioscience, San Diego, CA, USA) according to the manufacturer’s instructions. Single-cell suspensions from MLN and lung tissues were resuspended at 1 × 10⁶ cells/mL. The cells were labeled with the following monoclonal fluorochrome-conjugated antibodies: anti-CD3-Brilliant Violet 510 (clone 17 A2, BioLegend, San Diego, CA, USA), anti-CD4-eFluor 450 (clone RM4-5, eBioscience), anti-CD25-APC (clone PC61, BD Pharmigen), anti-OX-40-PE (clone OX-86, eBioscience), anti-PD-1-APC-eFluor 780 (clone J43, eBioscience), and anti-GITR-PE-Cyanine 7 (clone DTLA-1, eBioscience) for 30 min at 4°C in the dark. The cells were then washed in PBS and incubated with a fixation buffer (eBioscience) for 30 min at 4°C in the dark. After washing, the cells were resuspended in permeabilization solution 1× (eBioscience) and stained with anti-FOXP3-Alexa Fluor 488 (clone FJK-16s, eBioscience) for 30 min at 4°C in the dark. Flow cytometry was performed on a FacsCANTO II TM (BD, San Diego, CA, USA). At least 100,000 events were analyzed with FlowJo (Tree Star) or FACSDiva (BD Biosciences) software. Gating strategy is indicated in Figure S2 in Supplementary Material.

For cell surface staining, lymph node cells at a density of 2×10⁶ were first stained with 4 μg/mL anti-DTR-biotin (R&D cat.BAF259) and then stained with Streptavidin-PE (BioLegend cat.405204) along with either of the following fluorochrome-conjugated antibodies: anti-CD4-PerCP clone GK1.5 (Tianjin Sungene Biotech Co.), anti-CD44-APC clone IM7 (eBioscience), anti-B220-PerCP-cy5.5 clone RA3-6B2 (eBioscience), anti-CD62L-APC clone MEL-14 (BioLegend), and anti-CD8-Brilliant Violet 605 clone 53–6.7 (BioLegend). For intracellular Foxp3 staining, cells were first treated with the antibodies specific for cell surface and later stained with anti-Foxp3-Alexa Fluor488 clone FJK-16s (eBioscience) using the intracellular-staining kit from eBioscience. Cells were analyzed on Fortessa (BD Biosciences) or FACS Calibur (BD Biosciences). Data were analyzed using FACS analysis software (FlowJo).