Facs permeabilizing solution 2

Expression of intracellular IFNγ was determined in (subsets of) NK cells, γδ T cells, CD4⁺ and CD8⁺ T cells, using the assay adapted from Ariaans et al. [45 (link)]. Lymphocytes isolated from the IEL population and spleen were resuspended in complete medium, and 1 × 10⁶ lymphocytes in 0.5 mL were incubated in the presence of 1 µL/mL Brefeldin A (Sigma Aldrich) for 4 h at 41 °C, 5% CO₂. After incubation, lymphocytes were washed in PBA and stained as described in “Phenotypic characterization of lymphocytes by flow cytometry” section with surface markers as mentioned in the IFNγ panel (Table 1). Cells were washed in PBS, stained for viability and washed again in PBA. Then, lymphocytes were permeabilized differently as described by Ariaans et al. [45 (link)]. Lymphocytes were incubated in 200 µL of a mixture of BD FACS™ Permeabilizing Solution 2 and BD FACS™ Lysing Solution prepared according to manufacturer’s instructions (BD Biosciences) for 8 min at RT, immediately followed by centrifugation for 2 min at 393×g at 4 °C. Cells were washed twice in PBA, stained intracellularly with anti-IFNγ-APC in 50 µL PBA for 20 min at 4 °C in the dark, washed in PBA and finally analyzed by flow cytometry.

Peripheral blood drawn in vacutainer tubes supplemented with sodium citrate as anticoagulant or freshly prepared buffy coat were used for flow cytometry. The samples (whole blood or buffy coat) were stained with fluorochrome-conjugated mouse anti-human monoclonal antibodies (CD14-BV421, CD15-PE-Cy7 and CD3 PE-Cy5) from BD Biosciences (Franklin Lakes, NJ, USA). Mouse anti-human BSSL mAb AS20 was conjugated to Alexa Fluor 647 (AS20-AF647) using the Alexa Fluor® Antibody Labeling Kit (Molecular Probes by Life Technologies, Thermo Fisher Scientific), according to the manufacturer´s instruction, and used to detect BSSL. Exogenous biotin-labelled BSSL (bio-BSSL) was detected using BD Horizon BB515 Streptavidin (BD Biosciences). To analyze antigens on the cell surface, the protocol for direct immunofluorescence staining of whole blood using a lyse/wash procedure was used, as previously described (www.bdbiosciences.com). To analyze intracellular markers, the cells were first permeabilized using BD FACS™ permeabilizing solution 2 (BD Biosciences) before staining was performed following the manufacturer’s instruction. Flow cytometry was performed on a FACS LSR II (BD Biosciences) and data were analyzed using FlowJo software (BD Biosciences). Leukocyte populations were defined as CD14⁺ monocytes, CD15⁺ granulocytes and CD3⁺ T lymphocytes, respectively.

PBMCs (1.5 × 10⁶ cells/1.5 ml) were stained using two markers to identify pDCs: BDCA‐2‐FITC (MACS Miltenyi Biotec) and HLADR‐PE (MACS Miltenyi Biotec). The PBMCs were then incubated with IL4‐Ra‐APC (R&D Systems) or TLR‐7‐APC (Thermo Fisher Scientific) (Figure 1). The addition of fluorescence‐conjugated antibodies for flow cytometry was conducted in accordance was the manufacturer's protocol. TLR‐7 staining involved staining with BDCA‐2 and HLA‐DR followed by the fixing of PBMCs with BD FACS Permeabilizing Solution 2 (BD Biosciences) for 20 min and then resuspension with RPMI 1640 for further APC staining. A total of 3 × 10⁵ cells were acquired and analyzed on a BD FACScan flow cytometer (BD Biosciences) using CellQuest software.

For intracellular staining of IFNγ 1 × 10⁶ PBMCs were incubated with Phorbol 12-myristate 13-acetate (PMA) (50 ng/mL) plus ionomycin (500 ng/mL) for 4 h at 37 °C and with Brefeldin A (10 μg/mL) for the last 3 h, as previously described^{14 (link)}. Cells were first stained for surface antigen with CD3 PerCP antibody, fixed and after permeabilization with BD FACS permeabilizing solution 2 (BD Biosciences) stained for anti-IFN-γ PE (Becton Dickinson). Intracellular IFNγ is expressed as the percentage in PBLs, as well as in CD3 T lymphocytes.

At 0, 3, 7, 14 and 21 dpi, expression of intracellular IFNγ was determined in NK cells, γδ T cells, CD8⁺ T cells and CD4⁺ T cells, using an assay adapted from Ariaans et al. [50 (link)]. Briefly, lymphocytes isolated from the IEL population and spleen were suspended in complete medium, and 1 × 10⁶ lymphocytes in 0.5 mL were incubated in the presence of 1 µL/mL Brefeldin A (Sigma Aldrich) for 4 h at 41 °C, 5% CO₂. After incubation, lymphocytes were washed in PBA and stained as described above with surface markers summarized in the IFNγ panel (Table 1). Cells were washed in PBS, stained for viability and washed again in PBA. Next, lymphocytes were permeabilized differently as described by Ariaans et al. [50 (link)]. Here, lymphocytes were incubated in 200 µL of a mixture of BD FACS™ Permeabilizing Solution 2 and BD FACS™ Lysing Solution prepared according to manufacturer’s instructions (BD Biosciences) for 8 min at RT and immediately centrifuged for 2 min at 393 × g and 4 °C. Cells were washed twice in PBA, stained intracellularly with anti-IFNγ-APC in 50 µL PBA for 20 min at 4 °C in the dark, washed in PBA and finally analyzed by flow cytometry.