Monoclonal anti mouse cd3 pe cy5 labeled and cd4 fitc labeled antibodies

Additional female mice were used to analyze total and differential cell counts in BALF (n = 10/group). For this purpose, a polyethylene cannula was inserted into the trachea and a total volume of 1.0 mL of PBS containing 10 mM EDTA was instilled and aspirated (only once). BALF was centrifuged at 300 × g for 10 minutes at 4 °C. The supernatant was removed and the pellet resuspended in 250 μL PBS. Total leukocyte counts in BALF were quantitated in Neubauer chambers under light microscopy after dilution of the samples in Türk solution (2% acetic acid).
Cell suspensions from BALF were stained with monoclonal anti-mouse CD3 (Pe-Cy5-labeled) and CD4 (FITC-labeled) antibodies (eBioscience, San Diego, CA, USA) to assess CD3⁺CD4⁺ T-cell percentages. The regulatory T cell (Treg) population was characterized by staining for CD4, CD25, and Foxp3, as per manufacturer instructions (Mouse Treg Staining Kit, eBioscience, San Diego, CA, USA). Additionally, anti-mouse Siglec-F antibody (PE-labeled; BD Pharmingen, San Diego, CA, USA) was used to quantify eosinophils in the polymorphonuclear-gated populations in BALF samples. All data were acquired in a FACSCalibur flow cytometer (BD Biosciences Immunocytometry Systems, San Jose, CA, USA) and analyzed using FlowJo X 10.0.7 software (Tree Star Inc., Ashland, OR, USA).

Thymuses and mediastinal lymph nodes were removed from mice, placed into 1 mL PBS, and homogenized. Total cells in thymus and lymph nodes were counted in a Neubauer chamber after dilution in Türk solution. Cell suspensions from thymus and lymph nodes were stained with monoclonal anti-mouse CD3 (Pe-Cy5-labeled) and CD4 (FITC-labeled) antibodies (eBioscience, San Diego, CA, USA) to assess CD3⁺CD4⁺ T cell percentages. These data were acquired in a FACSCalibur flow cytometer (BD Biosciences Immunocytometry Systems, San Jose, CA, USA) and analyzed using FlowJo X 10.0.7 software (Tree Star Inc., Ashland, OR, USA).