Lsr ll

Freshly isolated or cultured PBMCs were stained with Zombie Aqua™ Fixable Viability Kit (BioLegend) for 20 min at room temperature and were incubated with anti-human FcR Blocking Reagent (Miltenyi Biotec, #130–059-901) and were then incubated with cell surface antibodies for 30 min on ice. The following monoclonal antibodies were used for phenotyping of freshly isolated PBMC: anti-CD3-APC-Cy7 (clone HIT3a, Biolegend, #300317), anti-CD4-APC (clone RPA-TA, Biolegend, #300514), anti-CD8- PerCP-Cy5.5 (clone SK1, Biolegend, #344709), anti-CCR4-PE-Cy7 (clone L291H4, Biolegend, #359409), anti-CCR7-FITC (clone G043H7, Biolegend, #353215), anti-CCR10-PE (clone 314305, R&D #FAB3478P-025), anti-CD45RO-BUV395 (clone UCHL1, BD Biosciences, #564292). And the following monoclonal antibodies were used for analyzing of proliferating lymphocytes within cultured PBMC: anti-CD3-APC-Cy7, anti-CD4-PE-Cy7 (RPA-TA, Biolegend, #300512), anti-CD8-BV421 (clone RPA-T8, Biolegend, #301036). Cells were acquired with an LSR ll (BD Biosciences) and data were analyzed by using FlowJo software (FlowJo, LLC, Ashland, OR, USA).

Cells were stained with Zombie Aqua Fixable Viability Kit (BioLegend) for 20 min at room temperature and were incubated with anti-mouse CD16/32 Antibody to block Fc receptors and were then incubated primary antibodies for 30 min on ice. For intracellular cytokine staining, cells were fixed and permeabilized using BD Cytofix/Cytoperm and were then incubated with anti-cytokine antibodies for 30 min on ice. For transcription factor staining, cells were fixed and permeabilized with Foxp3/Transcription Factor Staining Buffer Set (eBioscience) and were incubated with anti-transcription factor antibodies for 30 min on ice. The antibodies were purchased from BioLegend, eBioscience or BD Biosciences as detailed in the Key Resources Table. The data was acquired by LSR ll (BD Biosciences) and analyzed by using FlowJo software (FlowJo, LLC). Zombie Aqua-stained dead cells and doublet cells were removed from the analysis and CD45⁺ cells were gated as hematopoietic cells. To distinguish ILCs from other immune cells, lineage markers were used. Lineage markers included CD3e, CD5, CD19, CD11c, CD11b, FcεRIα, TCRγδ, TCRαβ, NK1.1 and B220. To exclude epidermal lymphoid cells, anti-CD2 antibody was included in the lineage markers except in experiments displayed in Figure 1A, Figure 5H, and Figure S5C. To sort cells for RNA-seq and ATAC-seq, 7-AAD was used for dead cell staining.