Anti human ifn γ pe cy7

Vγ9Vδ2 T cells were stimulated with 5 μg/mL plate-bound anti-CD3 antibodies and 1 μg/mL soluble anti-CD28 antibodies (free mAbs) for 6 h in the presence of 5 μg/mL Brefeldin A. Then, the cells were stained with anti-human CD3-APC-H7 (BD Biosciences, clone: SK7), anti-human TCR Vδ2-PerCP (BioLegend, clone: B6), and anti-human CD107a-APC (BD Biosciences, clone: H4A3) antibodies. After staining for surface markers, the cells were fixed and permeabilized using Lysing Solution (BD Biosciences) and Permeabilizing Solution (BD Biosciences), respectively. Subsequently, the cells were stained with anti-human IFN-γ-PE-Cy7 (BD Biosciences, clone: B27), anti-human TNF-α-PE (BD Biosciences, clone: MAb11), and their corresponding isotype controls (APC-conjugated mouse IgG1, κ isotype control (clone: MOPC-21); PE-Cy™7-conjugated mouse IgG1, κ isotype control RUO (clone: MOPC-21); and PE-conjugated mouse IgG1, κ isotype control (clone: MOPC-21), all from BD Biosciences). Then, the cells were analyzed with a BD FACS Verse, and the data were analyzed with FlowJo. In addition, cell cycle, cell proliferation (Ki-67, BioLegend, clone: Ki-67), cell differentiation (CD45RA, BioLegend, clone: HI100), CD27 (BioLegend, clone: O323), mitochondrial, and costimulatory molecule (CD80, BioLegend, clone: 2D10; CD86, BioLegend, clone: BU63) analyses were performed following standard protocols.

We obtained PBMCs from patients with high or low levels of TSLP by density centrifugation using Ficoll‐Paque and cultured these cells in RPMI 1640 supplemented with 10% FBS. Cells were resuspended at a density of 0.5 × 10⁵ cells·mL⁻¹, and 100–200 μL of cell suspension was stimulated with cell stimulation cocktail (plus protein transport inhibitors; 1.5 μL·mL⁻¹ of cell suspension; BD Biosciences). After stimulation in vitro, these cells were permeabilized with fixation and permeabilization buffer kit (BD Biosciences) and stained for 30 min at 4 °C in flow tubes with directly conjugated antibodies. The cells were fixed with 4% paraformaldehyde. The antibodies used for the flow cytometry analysis included anti‐human CD4‐allophycocyanin (560158; BD Biosciences), anti‐human IFN‐γ‐PeCy7 (557844; BD Biosciences), anti‐human IL‐4 (554516; BD Biosciences), anti‐human forkhead box P3 (Foxp3)‐Alexa Fluor 488 (560459; BD Biosciences) and anti‐human IL‐10‐phycoerythrin (554498; BD Biosciences). The cells were separated with a multicolor flow cytometer (BD Company, Franklin lakes, NJ, USA), and the fluorescence‐activated cell signaling data were analyzed by flowjo 10.0 software (BD Bioscience, San Jose, CA, USA).