Mouse igg1 kappa

Tetraspanin-like proteins in arthropod cells, including mosquito C6/36 cells, have been described previously [16 (link),30 (link),36 (link),37 (link),38 (link)]. Briefly, the C6/36 cells (mock and ZIKV-infected cells) were collected from the cell culture plates in sterile 1.5 mL microcentrifuge tubes, centrifuged at 550× g for 10 min at 4 °C, and separated from the medium. The cell pellets were fixed with 2% paraformaldehyde for 5 min at 4 °C, permeabilized with 0.1% Triton X-100 (Sigma) for 5 min at 4 °C, blocked for nonspecific binding sites with 2% BSA for 30 min at RT, and washed with 0.5% BSA. The cells were stained with the phycoerythrin (PE)-conjugated mouse anti-human CD63 antibody (Catalog #557305, BD Pharmingen) at a 1:20 dilution in 0.5% BSA and incubated for 1 h at RT with constant 1000 rpm agitation. The samples were suspended in 300 μL of 0.5% BSA and analyzed by using the FACSCalibur flow cytometer. The mouse IgG1 kappa (P3.6.2.8.1) antibody (Catalog #14-4714-82, eBioscience, San Diego, CA, USA) was used as an isotype control. Isotype FACS average values were rested from the mock and the infected cells FACS values.

After the aforementioned treatment, murine BMDM were digested and washed with PBS three times. Cells were collected and incubated with CD68 antibody (1:500, eBioscience, San Diego, CA, USA) or CD206 antibody (1:500, eBioscience, San Diego, CA, USA) for 30 min at room temperature. Mouse IgG1 kappa was used as negative control (eBioscience, San Diego, CA, USA). The ratio of CD68⁺ cells and CD206⁺ cells were analyzed using a flow cytometer (Thermo Fisher Scientific, Waltham, MA, USA).

U266 cells were incubated with 500 µM of HLA-A1 (VMAPRTLLL), HLA-B7 (VMAPRTVLL), or a non HLA-E binding control peptide (RGPGRAFVTI) (Biosynthesis Inc.) overnight at 37°C, 21% O₂ as previously described (15 (link), 18 (link)). Additional negative controls were included by incubating U266 cells in DMSO, the peptide’s solvent or in the medium. After the incubation, HLA-E expression was determined by flow cytometry by staining the cells with an HLA-E antibody (clone: 3D12 HLA-E, eBioscience) or with a matched isotype control, mouse IgG1 kappa (clone: P3.6.2.8.1, eBioscience). Following the induction, U266 cells were used in the CD107a assay (Figure 6) as described in the previous section.