Pe labeled anti cd25

Mouse Treg cells were collected from the BALF and analyzed for CD4⁺ CD25⁺ Foxp3⁺ expression using a mouse Treg cell staining kit containing APC-labeled anti-CD4, PE-labeled anti-CD25, and FITC-labeled anti-Foxp3 (eBioscience) according to the manufacturer’s instructions. Briefly, prepared cells (1 × 10⁶) were washed by centrifugation with cold PBS, resuspended in 1 mL of fixation/permeabilization solution, and incubated in the dark at 4°C for 30–60 min. The cells were washed once with 2 mL of permeabilization buffer, collected by centrifugation, resuspended in 20 mL of blocking agent with 2% (2 mL) normal rat serum in permeabilization buffer, and incubated at 4°C for 15 min. Next, 20 mL of a fluorochrome-conjugated antibody or isotype control in permeabilization buffer was added, followed by incubation in the dark at 4°C for 30 min. Finally, the cells were washed with 2 mL of permeabilization buffer, resuspended in flow cytometry buffer (PBS with 2% FBS), and analyzed using a FACSCanto II cytometer (BD Bioscience, San Diego, CA, United States). The data were analyzed using FlowJo^® software.

Blood samples from the adolescents were collected in a tube containing EDTA; 100 μl was transferred to tubes for monoclonal antibody labeling. Briefly, 100 μl of blood was incubated for 30 min at +4°C with antibodies specific for cell surface antigens (PE-labeled anti-CD4, APC-labeled anti-CD14, and FITC-labeled anti-CD16 for CD4 lymphocytes and monocytes M1 and M2, respectively) (eBioscience). In the other tube, 100 μl of blood was treated with fixation/permeabilization buffer (eBioscience) at +4°C for 40 min and after incubation washed three times with permeabilization buffer to allow intracellular staining with APC specific for FoxP3 antibody (eBioscience) at +4°C for 30 min. After one wash, the cells were incubated with PE-labeled anti-CD25 and FITC-labeled anti-CD3 (eBioscience) for 30 min at +4°C. For both staining procedures, appropriate isotype-matched controls were used. Acquisition (100,000 events) and analysis of cell populations were performed by flow cytometry (ACCURI, Becton & Dickinson) and presented as the mean fluorescence intensity (MFI).

Mouse Treg cells were collected from the BALF and analyzed for CD4+ CD25+ Foxp3+ expression using a mouse Treg cell staining kit containing APC-labeled anti-CD4, PE-labeled anti-CD25, and FITC-labeled anti-Foxp3 (eBioscience) according to the manufacturer’s instructions. Briefly, prepared cells (1 × 10⁶) were washed via centrifugation with cold PBS, resuspended in 1 ml of fixation/permeabilization solution, and incubated in the dark at 4 °C for 30–60 min. The cells were washed once with 2 ml of permeabilization buffer, collected via centrifugation, resuspended in 20 ml of blocking agent with 2 ml of 2% normal rat serum in permeabilization buffer, and incubated at 4 °C for 15 min. A fluorochrome-conjugated antibody or isotype control in 20-ml permeabilization buffer was added, followed by incubation in the dark at 4 °C for 30 min. The cells were washed with 2 ml of permeabilization buffer, resuspended in flow cytometry buffer (PBS with 2% FBS), and analyzed using a FACSCanto II cytometer (BD Bioscience, San Diego, CA, United States). The data were analyzed using the FlowJo® software.