Ebioscience fixation permeabilization concentrate

Polarized BMDMs or PMs were incubated in cold PBS containing 2 mM EDTA for 30 min, then the adherent cells were gently scraped off with a spatula and transferred to a centrifuge tube. After centrifugation at 500 g at 4°C, cells were blocked by the commercial Fc-receptors (anti-mouse CD16/32) (Biolegend, San Diego, USA) for 30 min at 4°C. Cell surface proteins were stained with indicated antibodies for 30 min, then, after fixation and lysis using eBioscience™ Fixation/Permeabilization Concentrate (Thermo Fisher Scientific, Waltham, USA) for 30 min; intracellular proteins were stained for 30 min.
For mouse tumor tissues, they were cut into pieces and digested with collagenase IV (2 mg/mL, Sigma-Aldrich, Taufkirchen, Germany) and DNAase I (20 U/mL, Sigma-Aldrich, Taufkirchen, Germany) for 30 min in a 37°C incubator and filtered through a 70 μm cell strainer to obtain a single cell suspension. After lysing the red blood cells using RBC lysis buffer, the obtained cells perform blocking and staining as described above.
All cells samples were resuspended in PBS and analyzed using a flow cytometer (Leica, Wetzlar, Germany) and Flow Jo (v10.6.2).

Detection of HEV ORF2 Ag in the infected cells was done by flow cytometry as described previously [18 (link)]. Briefly, HMOs and HMACs were challenged with the stool-derived HEV-1 and/or HEV-3 as described; the cells were fixed at day 12 post infection and permeabilized using eBioscience™ Fixation/Permeabilization Concentrate (ThermoFischer Scientific, USA) according to the manufacturer’s instruction and then stained with antibody 1E6 clone (Millipore, Billerica, MA, United States; dilution 1/1000) that targets the HEV ORF2 protein. Goat anti-mouse IgG conjugated with Alexa488 (Invitrogen, Waltham, MA, USA) was used as a secondary antibody according to the manufacturer’s instructions. For detection of ds-RNA, the cells were fixed using cold methanol, permeabilized by 0.5% Triton X100, and stained with mouse monoclonal anti-dsRNA J2 Ab (Millipore; dilution 1/200). Goat anti-mouse IgG conjugated with Alexa647 (Invitrogen) was used as a secondary antibody (dilution 1/1000), and DAPI was used for nuclei staining.

PHESCs cells were challenged with the stool-derived HEV-1 and/ or HEV-3 as described in the previous section. After 14 days, the cells were fixed using cold methanol, permeabilized by 0.5% Triton X100 and stained with antibody 1E6 clone (Millipore; dilution 1/500 for IF). Goat anti-mouse IgG conjugated with Alexa647 (Invitrogen) was used as a secondary antibody (dilution 1/2000 for IF) and DAPI was used for nuclei staining. For flow cytometry, the cells were fixed and permeabilized using eBioscience™ Fixation/Permeabilization Concentrate (ThermoFischer Scientific, CA, USA) according to the manufacturer’s instruction and then stained with antibody 1E6 clone (Millipore; dilution 1/1000) that targets the HEV ORF2 protein. Goat anti-mouse IgG conjugated with Alexa488 (Invitrogen) was used as a secondary antibody according to the manufacturer’s instructions. Uninfected PHESCs processed by the same procedure were used as a negative control.