Facscelesta instrument

Tumor tissue was minced, digested with collagenase IV (Sigma), and incubated with RBC lysis buffer (BD Biosciences) to lyse the red blood cells. The resulting single‐cell suspension was washed and resuspended in phosphate‐buffered saline (PBS) containing 0.1% BSA. Subsequently, membrane markers were stained with fluorochrome‐conjugated antibodies and incubated at 4 °C for 40 min. The single‐cell suspension was also stained for intracellular proteins using corresponding fluorochrome‐conjugated antibodies and the Fixation/Permeabilization Solution Kit (BD Biosciences). FACS data were collected using a BD FACS Celesta instrument and analyzed using FlowJo V.10.0. Detailed information regarding the FCM antibodies is provided in Table S2 (Supporting Information).

In order to detect the expression of IFN-γ and IL-4 in transfected naive CD4⁺ T cells after immunization, the cells were surface stained with PerCP-Cyanine5.5-anti-CD3 (Thermo Fisher Scientific Co., Ltd, Xian, China) and APC-anti-CD4 antibodies (Thermo Fisher Scientific Co., Ltd, Xian, China). Next, the cells were treated with fixation buffer (BioLegend, Inc, California, USA.) and permeabilization buffer (BioLegend, Inc, California, USA.) followed by intracellular staining with fluorochrome-conjugated PE-anti-IFN-γ (Thermo Fisher Scientific Co., Ltd, Xian, China) and PE-anti-IL-4 (Thermo Fisher Scientific Co., Ltd, Xian, China). When assaying CD4⁺ T cells from immunized mice, staining is performed using PerCP-Cyanine5.5-anti-CD3 (Thermo Fisher Scientific Co., Ltd, Xian, China), APC-anti-CD4 antibodies (Thermo Fisher Scientific Co., Ltd, Xian, China), PE-anti-IFN-γ (Thermo Fisher Scientific Co., Ltd, Xian, China), and BV605-anti-IL-4 (Thermo Fisher Scientific Co., Ltd, Xian, China). The degree of cell activation after transfection has been determined by staining the surface of naive CD4⁺ T cells with PE-anti-CD25 and FITC-anti-CD69 antibodies (Thermo Fisher Scientific Co., Ltd, Xian, China). The stained cells were measured by a BD FACSCelesta instrument (Beijing, China) and the results were analyzed by FlowJo.

Cell pellets were washed with PBS, fixed with 4% paraformaldehyde and permeabilized with methanol at −20°C. Cells were stained for one hr with Phospho-Rb Ser 807/811 (D20B12 XP, Cell Signaling Technology, Danvers, MA) at 1:500 dilution prior to Alexa Fluor 488 secondary antibody (ab150073, Abcam, Cambrdige, MA) staining at 1:500 dilution for one hr. Antibodies were diluted in PBST with 1% BSA. PI DNA staining was performed using FxCycle PI/RNAse (Thermo Fisher Scientific F10797). FACS for phospho-Rb and PI was performed on a BD FACSCelesta instrument and analyzed with BD FACSDiva v8 software (BD Biosciences, San Jose, CA). GFP: Ex 488, Em 530/30; mCherry Ex 561, Em 610/20. FACS enrichment of stable cell lines was performed using BD FACSAria Fusion with the following optics: CFP: Ex 445, Em 470/15; YFP: Ex 488, Em 530/30; and mCherry Ex 561, Em 610/20.

The ear single-cell suspension was washed two times with PBS. First, cells were adjusted to a concentration of 1 × 10⁶ in 200 μL PBS and stained with Zombie fixable dyes for 20 min at room temperature. Later, cells were washed with 1 mL of FACS buffer, resuspended in 100 μL of FACS buffer, and incubated with Fc receptor-blocking antibodies for 15 min at 4°C followed by surface staining with conjugated antibodies for 25 min at 4°C. The complete list of antibodies used in this study is described in Table S1 in the supplemental material. Stained cells were washed with FACS buffer and filtered through 30-μm filters, and data were acquired with the BD FACSCelesta instrument. Data were analyzed using FlowJo v10.8 Software (BD Life Sciences) (79 ).

Briefly, individual human lymphocyte samples were thawed, washed, and incubated for 30 min at 4 °C in 1× PBS with 10% normal mouse serum (Beijing Yaanda Biotechnology). The cells were then stained with antibodies for 30 min at 4 °C in the dark. Then the cells were washed with MACS buffer and incubated with the dead cell marker 7-AAD. A FACSAria cell sorter (BD) was used to purify ILCs[Lin^- (CD34^-CD3^-CD14^-CD1α^-CD19^-FcεRIα^-) CD45⁺]. And a FACSCelesta instrument (BD) was used to detect NK cells. The details regarding the antibodies are shown in the Supplementary materials and methods (Doc S1).

16HBE14°− cells were seeded at a density of 1 × 10⁵ cells/well in 12-well plates in duplicate wells per experimental condition. After treatment, cells were washed, detached with 0.05% Trypsin-EDTA, placed in FACS tubes and fixed with 4% PFA at room temperature for 15 min. Cells were washed with PBS and permeabilised by washing twice in PBS with 0.05% saponin. After a blocking step in 5% goat serum, primary antibodies were left overnight at 4°C in PBS with 0.05% Saponin and 5% goat serum. Fluorochrome conjugated secondary antibodies were added for 1 h at RT, and after final washing, cells were acquired in a FACS Celesta instrument (BD Biosciences).