Propidium iodide

Naive CD4⁺ T helper cells were isolated from the spleen of BALB/c mice using FACS Aria II (BD Bioscience). Red blood cells were removed from the splenic suspension using ACK buffer and splenocytes were stained with Alexa Fluor 700 labeled anti-CD4 mAb (GK1.5; BioLegend), dead cells were excluded by propidium iodide (Exbio, Vestec, Czech Republic). J774 cells (2 × 10⁴ cells/ml) were cultivated for 4 hours with or w/o stimulation. Then CD4⁺ T helper cells (2 × 10⁵ cells/ml) were added together with stimulation with LPS or capsaicin. After 72 h, the proliferation expression of Ki67 was detected using flow cytometry. Briefly, cells were harvested, washed with PBS/0.5% BSA and incubated for 30 min on ice with Alexa Fluor 700 labeled anti-CD4 mAb (GK1.5; BioLegend), APC labeled anti-CD11b mAb (M1/70; BioLegend) and LIVE/DEAD™ Fixable Violet Dead Cell Stain Kit (Thermo Fisher). Cells were then fixed and permeabilized using a Foxp3 Staining Buffer Set (Thermo Fisher) according to manufacturer’s instructions. Subsequently, cells were stained intracellularly for 30 min with PE labeled anti-Ki67 mAb (SolA15; Thermo Fisher). All events were analyzed after the exclusion of dead cells and debris. Data were collected using the LSR II cytometer and analyzed using GateLogic 400.2 A. Representative dot plots illustrating the gating strategy are shown in Supplementary Fig. S4.

Cell apoptosis was measured after staining with annexiv V and propidium iodide (exbio), as per manufacturer’s instructions. In brief, cells were washed with PBS and were incubated with annexiv V and propidium iodide for 15 min. After incubation, they were washed, resuspended in buffer, and analyzed by FCM.