Cytoflex s analyzer

CD3⁺ cells were depleted from the freshly thawed PBMCs using EasySep Positive Selection Kits II (Stemcell). Flowthroughs consisting of CD3⁻ cells were collected. The cells (5 × 10⁵) were then pulsed with 5 μg/ml of peptides or DMSO for 1 h in 37°C. After incubation, pulsed cells were washed twice before addition of autologous nasal cells together with anti-CD40L-PE (RRID: AB_314828; BioLegend) and anti-CD107a-APC (RRID: AB_1727417; BD). After 3 h of incubation at 37°C, the cells were washed in PBS stained with Zombie NIR Fixable Viability Kit to exclude dead cells in subsequent analysis. Then, they were washed in FACS buffer and stained with surface markers anti-CD3-BV605 (RRID: AB_2561911; BioLegend), anti-CD4-BV650 (RRID: AB_2744425; BD), anti-CD8-PE-Cy7 (RRID: AB_396852; BD), anti-CD69-AF700 (RRID: AB_493775; BioLegend), and anti-CD103-FITC (RRID: AB_10597744; eBioscience) diluted in FACS buffer for 30 min on ice. After two more washes in FACS buffer, cells were resuspended in PBS before acquisition with a Beckman Coulter CytoFLEX S analyzer.

All antibodies used are listed in Supplementary Table 1. Stained samples were acquired on a CytoFLEX S analyzer (Beckman Coulter), and analysis was performed using FlowJo v10 (TreeStar).

Isolation and staining plant nuclei were performed by using a flow reagent kit CyStain PI Absolute P (Sysmex Europe GmbH, Norderstedt, Germany). Young leaves were thoroughly chopped using a razor blade in the presence of 1 mL extraction buffer (200 mM Tris, 4 mM MgCl₂·6H₂O, 0.5% Triton X-100, pH 7.5) and then filtered through a 50 μm disposable filter. For staining of nuclei, 50 μL propidium iodide staining solution and 5 μL RNase A were added to the tube and mixed well. The tube was incubated on ice for 1 h. The nucleus solution of each sample was analyzed for ploidy level by a flow cytometer (CytoFLEX S Analyzer, Beckman Coulter, Brea, CA, USA). Chicken erythrocyte nuclei (CEN) (BioSure, Grass Valley, CA, USA) were used as an internal biological reference in flow cytometry. The published genome size of CEN is 2.5 picograms (pg) (1 pg = 10⁻¹² g) [42 ]. The diploid genome size of W. chinensis was determined to be 4.5 pg, as revealed by flow cytometry analysis.