2.3. Flow Cytometry

To detect FLAG-IFNLR1 receptor expression on the cell surface, cells were fixed with fresh 4% paraformaldehyde (PFA; 15710, Electron Microscopy Sciences, Hatfield, PA, USA) for 20 min at 4 °C and stained with rat anti-DYKDDDK Alexa Fluor 488 (1:500, 637317, BioLegend) or rat IgG2a Alexa Fluor 488 Kappa isotype control (1:500, 400525, BioLegend) for 45 min at 4 °C. To detect intracellular receptor expression, cells were fixed and permeabilized using BD Cytofix/Cytoperm Fixation/Permeabilization solution kit (BDB554714, BD, Franklin Lakes, NJ, USA) prior to staining.

To detect pSTAT1, cells were harvested, fixed with 4% PFA for 20 min at 4 °C, and permeabilized in chilled 100% methanol (A411-4, Fisher Scientific) for 15 min at 4 °C. Cells were then stained with mouse anti-Stat1-PE (pY701) (5 µL/100 µL sample, 612564, BD Phosflow) prior to flow cytometry. Mock treated and non-permeabilized cells were used as negative controls.

Flow cytometry was performed using the Guava HT8 Incyte System (Luminex, Austin, TX, USA). Live cells were identified using scatter-area (forward and side scatter) and by staining with a viability dye (1:1000, Zombie Green Fixable Viability, 423111 or 1:1000, Zombie Red Fixable Viability, 423109, BioLegend). FlowJo software was used for analysis.