Pe conjugated anti icos

Briefly, at most 8 × 10⁶ CD4⁺-T-cells were surface stained with 5 µL peridinin-chlorophyll-protein-Cy5-5 (PerCpCy5.5)-conjugated anti-CD127 (eBioscience, Frankfurt, Germany), 20 µL phycoerythrin (PE)-conjugated anti-ICOS (BD Biosciences, Heidelberg, Germany), 5µL allophycocyanin-H7 (APC-H7)-conjugated anti-CD45RA (BD Biosciences), and 5 µL phycoerythrin-cyanine 7 (PE-Cy7)-conjugated anti-CD31 (eBioscience) mouse monoclonal antibodies. Intracellular staining for the detection of FoxP3 was performed using a fluoresceinisothiocyanat (FITC)-conjugated anti-human FoxP3 staining set (clone PCH101, eBioscience) according to the manufacturer’s instructions. Detection of Ki67⁺ cells within the different Treg/Tresp subsets was performed by incubating the fixed cells with 2 µL Alexa-flour 647-conjugated anti-Ki67-conjugated mouse monoclonal antibodies (clone B56, BD Biosciences). Negative control samples were incubated with isotype-matched antibodies. FSC-H versus FSC-A and FSC versus SSC gating was used for doublet and debris discrimination (for gating strategy see Figure S1). Cells were analyzed by a FACS Canto flow cytometer (BD Biosciences). Statistical analysis was based on at least 100,000 CD4⁺-T-cells.

APC-conjugated antilineage markers, PE-conjugated anti-ICOS, FITC-conjugated anti-CD90.2, and PE-Cy7–conjugated anti-T1/ST2 were obtained from BD Pharmingen (no. 558074) and eBioscience (no. 12-9949-81, no. 11-0903-82, and no. 25-9335-82). Flow cytometry was performed using BD FACSAria. ILC2 are defined as Lin(CD3, CD11b, CD45R/B220, Ly-76, Ly6G, and Ly-6C)-, Thy1.2⁺ICOS⁺T1/ST2⁺ cells. Data were analyzed using FlowJo software v.10 (TreeStar Inc.). Percentages of Lin-Thy1.2⁺ICOS⁺T1/ST2⁺ cells and total cells recovered from the lungs were used to determine the number of ILC2 cell recovery. For all analyses, isotype control staining was subtracted from true antibody staining to determine the percentage of positive cells.