Flow cytometry reagents

Flow cytometry reagents were all supplied by BioLegend (San Diego, CA, USA). 1 × 10⁶ splenocytes were incubated with 10 µg/mL protein gE at 37 °C in 5% CO₂ for 2 h to stimulate the expression of antigen-specific cytokines, and 5 µg/mL brefeldin A was then added and incubated overnight under the same conditions to block cytokine release. After washing with staining buffer, 100 μL of Zombie NIR™ was added to each well and incubated for 30 min to determine the proportion of dead cells in the sample. Then, 5 µg/mL anti-CD16/CD32 antibodies was added and incubated at 4 °C for 10 min to block nonspecific binding to Fc receptors. PerCP-Cy5.5-conjugated anti-mouse CD4, FITC-conjugated anti-mouse CD8, BV510-conjugated anti-mouse CD44, and BV421-conjugated anti-mouse CD62L antibodies were subsequently added and incubated for another 30 min at 4 °C for surface maker staining. PE-conjugated anti-mouse IFN-γ and APC-conjugated anti-mouse IL-2 antibodies were used for intracellular staining. After staining, live cells were gated (forward and side scatter, FSC/SSC), and acquisition with more than 20,000 events for CD4⁺ or CD8⁺ T cells in each sample were analyzed with a CytoFLEX flow cytometer (Beckman, Indianapolis, IN, USA) and FlowJo_V10 software (BD, Franklin Lakes, NJ, USA).

Auricularia polytricha mushroom was purchased from Chang Daeng mushroom farm in Samutprakarn province, Thailand. Dulbecco’s Modified Eagle’s Medium (DMEM), Roswell Park Memorial Institute (RPMI)-1640 medium, phosphate buffered saline (PBS), trypsin 0.25% (v/v), and antibiotics were purchased from HyClone, Utah, USA. Fetal Bovine Serum (FBS), 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), ergosterol, and propidium iodide were purchased from Sigma-Aldrich, Darmstadt, Germany. Hexane, ethanol, and dimethyl sulfoxide (DMSO) were purchased from RCI Labscan, Bangkok, Thailand. The flow cytometry reagents were purchased from BioLegend, California, USA. The human cytokine ELISA kits (IL-6, IL-1β, and TNF-α) were purchased from Thermo Scientific, Massachusetts, USA. Primary and secondary antibodies for Western blot analysis were purchased from Cell Signaling Technology, Massachusetts, USA. Bradford protein assay was purchased from Bio-Rad Laboratories, USA. Matrigel® and transwell® inserts were purchased from Corning, Arizona, USA.