Cytosoft software version 4

Splenocytes were isolated from mice as previously described^{32 (link)}. Briefly, RBCs were treated with lysis solution for 15 min at room temperature, after which 1 × 10⁶ cells were collected for analysis. For T cell activation, cells were incubated at 37 °C in RPMI1640 medium (GIBCO, Grand Island, NY, USA) supplemented with a cell activation cocktail containing brefeldin A (423303; Biolegend, San Diego, CA, USA) according to the manufacturer’s protocol. The activated cells were washed in phosphate-buffered saline (PBS) and incubated in 5% BSA/PBS for 15 min on ice. Immunostaining was then performed using anti-CD4 Alexa Fluor 488-conjugated antibodies (1:500; 100425; Biolegend). After 3 washes with ice-cold staining buffer, cells were resuspended in fixation buffer (420801; Biolegend), incubated for 15 min on ice, and washed. After permeabilization, cells were immunostained with PE/Cy7 anti-IFN-r (1:500; 505825; Biolegend) and PE/Cy7 anti-IL-4 (1:500; 504117; Biolegend) antibodies. Splenocyte staining was analyzed on a Guava EasyCyte mini instrument and data were analyzed using Cytosoft software version 4.2.1 (Merck Millipore, Billerica, MA, USA).

Cells were incubated with 20 μM of 2′,7′-Dichlorofluorescin diacetate (Sigma) in culture media for 20 minutes and detached with trypsin and collected in 1 mL of PBS. Cells were washed two times with 500 μL PBS and analyzed on a Guava EasyCyte mini instrument using Cytosoft software version 4.2.1 (Merck Millipore, Billerica, MA, USA).