Zombie green fixable viability

The following fluorescent dye-conjugated antibodies or reagents were used to label cells: FITC-CD8 (Clone RPA-T8, Cat:301,050), PerCP-Cy5.5-CD25 (Clone BC96, Cat:302,625), PE-HLA-DR (Clone L243, Cat:307,605), Alexa Fluor 488-CCR7 (Clone G043H7, Cat:353,205), APC/Cy7-CD45RA (Clone HI100, Cat:304,127), APC-CD122 (Clone TU27, Cat:339,007), PE-CD95 (Clone DX2, Cat:305,607), PE-CD19 (Clone HIB19, Cat:302,208), APC-TCRα/β (Clone IP26, Cat:306,718), FITC-HLA-A, B, C (Clone W6/32, Cat:311,404) and Zombie Green Fixable Viability (all from BioLegend, USA).
For the detection of cytokines-expressing cells, mice tumors were firstly digested with collagenase (Sigma-Aldrich, USA). The infiltrating mononuclear cells were obtained using the tumor lymphocyte isolation kit (Solarbio, China). The Intracellular (IC) Fixation buffer (eBioscience, USA) was added into cell medium and incubated at room temperature for 2 h to fix cells. After centrifugation for 5 min, cells were resuspended in permeabilization buffer (eBioscience, USA) and incubated with PE-IFN-γ and Alexa Fluor 700-Granzyme-B antibodies (BioLegend, USA) at 4 ℃ for 20 min. All flow cytometry data were acquired on FACS Aria II flow cytometer (BD Biosciences, USA) and analyzed using FlowJo software.

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.