Fixation permeabilization concentrate

We collected the cells and washed them with PBS. Then the cells were fixed with a fixation/permeabilization concentrate (Thermo Fisher Scientific, #00-5123-43) at 4°C for 0.5 h and permeated with a permeabilization buffer (Thermo Fisher Scientific, #00-8333-56) at 4°C for 5 min. Then the cells were incubated with Ki-67 APC direct-labeled antibody (Thermo Fisher Scientific, #17-5699-42) at room temperature for 20 min and washed with PBS. Flow cytometry was performed on the FACSymphony™S6 (BD Biosciences) platform, and results were analyzed using FlowJo software version 10.5.3.

Apoptosis of cells was detected by Annexin V staining (Yeason, China). After being extracted from the bone marrow of mice, 5 × 10⁶ cells were labeled with different surface markers for 30 to 45 min at 4 °C and then twice rinsed with PBS. Subsequently, the cells were reconstituted in binding buffer and supplemented with Annexin V. After 30 min of incubation, flow cytometry was detected in the FITC channel. Cell cycle analysis was performed with the fluorescein Ki-67 set (BD Pharmingen, USA), following the directions provided by the manufacturer. Briefly, a total of 5 × 10⁶ bone marrow cells were labeled with corresponding antibodies, as previously stated. Afterward, the cells were pre-treated with a fixation/permeabilization concentrate (Invitrogen, USA) at 4 °C overnight and subsequently rinsed with the binding buffer. The cells were stained with Ki-67 antibody for 1 h in the dark and then with DAPI (Invitrogen) for another 5 min at room temperature. Flow cytometry data were collected by a flow cytometer (CytoFLEX LX, Beckman, USA).

NK cells were collected and resuspended on cold PBS including 2% FBS. After that, the suspension was added to a 6-well plate and staining by APC anti-human CD107a (LAMP-1) (BioLegend, 328619, USA) and incubated for 1 hour at 4°C in the dark. Golgiblock (brefeldin A solution (1,000x) (Invitrogen, 00-4506-51, China) and monensin solution (1,000x) (Invitrogen, 00-4505-51, China)) was added into the cell suspension and cultured for 4 hours at incubator. After centrifugation and washing, the suspension was added into fixing agent (fixation/permeabilization concentrate (Invitrogen, 00-5123-43, China): fixation/Permeabilization diluent (Invitrogen, 00 − 5223 − 56, China) = 1 : 3) and incubated for 1 hour at 4°C in the dark. The cells were punched using Permeabilization Buffer (Invitrogen, 00-8333-56, China). Following centrifugation and washing, PE anti-human IFN-γ (BioLegend, 506506, USA) was added into the cell suspension overnight at 4°C. Flow cytometry readings were performed on the FACS.

An aliquot of the T cell-enriched samples from each donor was stained with anti-human CD3 (cat No. 300426, clone UCHT1, BioLegend, San Diego, CA, USA) for 30 minutes at room temperature to validate the isolation of T cells. The aliquots were also stained with anti-human CD3 (cat No. 300426, clone UCHT1, BioLegend), anti-human CD8 (cat No. 560662, clone RPA-T8, BD Biosciences, Franklin Lakes, NJ, USA), and anti-human CD45RO (cat No. 562299, clone UCHL1, BD Biosciences). The cells were then treated with fixation/permeabilization concentrate (cat No. 00-5123-43, Invitrogen, Waltham, MA, USA) to fix and permeabilize cells for 1 h at 4°C, and intracellular staining was conducted according to the manufacturer’s recommendations. The cells were stained with anti-human interferon γ (IFN-γ) (cat No. 56-7319-41, clone 4S.B3, Invitrogen), anti-human tumor necrosis factor α (TNF-α) (cat No. 25-7349-41, clone MAb11, Invitrogen), and anti-human IL-2 (cat No. 500307, clone MQ1-17H12, BioLegend). Sample data were acquired using the LSR Fortessa X20 and were analyzed using FlowJo software.

The eBioscience™ Cell Stimulation Cocktail (lot no.: 2046941) was purchased from Thermo Fisher Scientific Inc. (USA). FITC-conjugated anti-human CD4 (lot no.: B262223), PerCP-Cy5.5-conjugated anti-human CD3 (lot no.: B560835), Brilliant Violet 510-conjugated anti-human IFN-γ (lot no.: B323449), PE-conjugated anti-human IL-17A (lot no.: B315520), APC anti-human FOXP3 (lot no.: 1995356), PE-conjugated anti-human CD25 (lot no.: 2173867), Brilliant Violet 421-conjugated anti-human CD274 (lot no.: B320732), PE/Cyanine7-conjugated anti-human CD279 (lot no.: B318888), Fixation/Permeabilization Concentrate (lot no.: 2084746), eBioscience™ Fixation/Perm Diluent (lot no.: 2047346), Permeabilization Buffer 10× (lot no.: 2060497), and FIX&PERM (Fixation Medium A, Permeabilization Medium B) (lot no.: 17159) were purchased from Biolegend (USA).

At 3 dpi, HIE monolayers were harvested and separated into a single cell suspension by using Accumax (STEMCELL technologies, USA). HIE were kept in Advanced DMEM/F-12 (Gibco) supplemented with 2 mM of EDTA and sequentially stained with LIVE/DEAD Fixable Aqua Dead Cell Stain (Thermo Fisher Scientific) and (when indicated) with surface markers: CD44-BV421, Mucin-2-FITC, Sucrose-isomaltase-PE, CGA-PerCP and Lysozyme-APC. Cells were next fixed with Fixation/Permeabilization Concentrate (eBioscience) and kept in permeabilization buffer for intracellular staining either with i) the biotin-conjugated primary dsRNA (J2, Scicons) followed by the secondary streptavidin APC-Cy7-antibody, or ii) the primary antibodies dsRNA (J2, Scicons) and VA1-VP1 followed by the secondary anti-mouse AlexaFluor 488 and anti-rabbit AlexaFluor 564, respectively. For the surface marker, single color controls were obtained by staining BJAB B cells with CD45 labelled with BV421, FITC, PE, PerCP and APC whereas the intracellular staining was based on VA1-infected Caco-2 cells. Samples were acquired with BD Fortessa and analysed by FlowJo. For infection studies, the gate on the mock-infected control samples was set to ≤1% of positive events. Gates for the surface markers were set using undifferentiated HIE data as reference.