Ebioscience foxp3 transcription factor buffer set

Single-cell suspensions were prepared as described and incubated with Live/Dead Fixable Blue Dead Cell Stain (ThermoFisher) in PBS for 10 min at room temperature followed by blocking with anti-mouse CD16/CD32 (2.4G2) (UCSF Hybridoma Core Facility) and 5% normal rat serum for 10 min at room temperature. Cells were then washed in FACS buffer and stained for surface markers for 20 min at room temperature. For intracellular staining, cells were fixed and permeabilized using the eBioscience FoxP3 Transcription Factor Buffer Set (ThermoFisher) according to the manufacturer’s instructions. Cells were either sorted directly into DMEM (ThermoFisher) containing 10% FBS using a BD FACS Aria Fusion (BD Biosciences) or analyzed using a BD LSR II (BD Biosciences) housed within the UCSF Single Cell Analysis Center. Flow cytometry data was analyzed using BD FACSDiva v8.0 or FlowJo v10.5.3 software (TreeStar Software). The following antibodies were used in this study: Ly51-PE (6C3, BioLegend), CD11c-PE-Cy7 (N418, eBioscience), CD45-PerCP (30-F11, BioLegend), EPCAM-APC-Cy7 (G8.8, BioLegend), Aire-e660 (5H12, eBioscience), Ki67-PE (eBioscience), CCL21 (59106, R and D Systems), Goat anti-Rat IgG-A488 (ThermoFisher).

Peripheral blood was collected using a BD Vacutainer® blood collection tube containing EDTA and immediately sent for flow cytometry analysis. For flow cytometry, a 200‐μL aliquot of whole blood was stained with a cocktail of surface markers, which was selected mainly derived from the standardized phenotyping panel by the Human Immunophenotyping Consortium
²² (link) (the antibodies included anti‐CD3, anti‐CD4, anti‐CXCR3, anti‐CCR4, anti‐CCR6, anti‐CCR7, anti‐CD45RA, anti‐CD25, and anti‐CD127). Then, the mixture was lysed using red blood cell lysing buffer (TIANGEN). Subsequently, cells were fixed and permeabilized using eBioscience Foxp3/Transcription Factor Buffer Set (Thermo Fisher Scientific) and stained with anti‐EOMES antibody (clone: WD1928; Thermo Fisher Scientific) according to the manufacturer's instructions. Appropriate fluorescein‐conjugated, isotype‐matched, irrelevant antibodies were used as isotype controls. The flow cytometry experiments of the derivation cohort were performed using Attune NxT Flow Cytometer (Thermo Fisher Scientific) and analyzed on FlowJo VX Software (FlowJo, LLC). The flow cytometry experiments of the validation cohort were performed using BD FACSCelesta™ Flow Cytometer (BD Life Sciences).