Pe labeled anti cd4

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).

The following antibodies were used for flow cytometry studies: APC-labeled anti-F4/80 and FITC-labeled anti-CD11b, FITC-labeled anti-CD3 and PE-labeled anti-CD8, or FITC-labeled anti-CD3 and PE-labeled anti-CD4, PE-labeled anti-CD11C, and APC-labeled anti-Siglec-F, PerCP/Cy5.5-labeled anti-CD80, PE-Cy7-labeled anti-CD86, and PE-Cy7-labeled anti-MHC-II (eBioscience, San Diego, CA, USA; Thermo Fisher Scientific, Inc., Waltham, MA, USA) were used. To identify ILC2, lung cells were stained with PE-Cy7-labeled CD45, APC-labeled CD90.2, PE-labeled ST2, and FITC-labeled Lineage cocktail (anti-CD3, CD11b, B220, Gr-1, and TER119, eBioscience). The following antibodies were used for purification of eosinophils: APC-conjugated anti-CD19, anti-CD90.2, and anti-CD8α antibodies (BioLegend, San Diego, CA, USA) and APC-conjugated magnetic beads (MACS; Miltenyi Biotec, Auburn, CA, USA). Rabbit polyclonal antibody to major basic protein (MBP) was purchased from Affinity Biosciences (Changzhou, China) for immunohistochemical staining. OVA (A5503) and LPS (L2880, Escherichia coli 055:B5) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Aluminum hydroxide (Inject Alum, 77161) was purchased from Thermo Fisher. Human RSV type A (A2 strain) was propagated in A549 cells as previously described (36 (link)).