Propidium iodide staining

For lignin and structural carbohydrate quantifications and gene expression analysis, A. tequilana leaves samples were collected and processed as described immediately. Adult leaves (third full-expanded leaf) were collected from 4-year plants in Rancho Agropecuaria Santa Genóveva, Campeche México (19°33′03.0″ N 90°01′10.9″ W). On the lab-bench, three types of samples were obtained from leaves: pooled samples of the whole leaf, cells surrounding fibers, and fibers. For pooled samples of the whole leaf, three fractions (16 cm²) were dissected from the basal (C), medium (B), and distal (A) parts, frozen with liquid nitrogen (N₂), and kept at −80 °C until processing. These three leaf fractions were used to get a landscape of the whole leaf, as reported previously in Agaves [9 (link)]. For isolation of cells surrounding fibers, leaves were longitudinally segmented with a sterilized knife, and then fibers were detached one by one and immediately frozen with N₂ and kept at −80 °C until processing. The presence of surrounding cells still to fibers was verified by propidium iodide staining (0.2 μg ul⁻¹) (Sigma-Aldrich, St. Louis, MO, USA) and visualized by laser scanning confocal microscopy (514 nm line for excitation, Carl Zeiss, Jena, Germany). Fibers isolated after grounding with N₂ were washed deeply with tap water and dried to 65 °C.

Fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated monoclonal antibodies (mAbs) against the following DC markers were used: CD11c, CD80, CD86, PD-L1, PD-L2 or HLA-ABC (BD Pharmingen, San Diego, CA, USA); CD14, CD40, CD83, and HLA-DR (eBioscience, San Diego, CA, USA); and CD197 (R&D Systems). All analyses were performed on a FACSCalibur flow cytometer (BD Biosciences). After staining cells with each antibody, dead cells were removed by propidium iodide staining (Sigma-Aldrich, Steinheim, Germany), and live cells gated on forward scatter (FSC) and side scatter (SSC) without the lymphocyte population were examined for immunophenotyping.

Human blood leukocytes viability after collection was used as an indicator of blood degradation over time. Whole blood was collected and incubated for up to 8 h at 37 °C and 80 rpm (PSU-10i, Biosan). At time points 0 (immediately after collection), 4 h and 8 h, two mL of whole blood was collected and incubated with five mL of red blood cells (RBC) lysis buffer (Alfa Aesar, Karlsruhe, Germany). The suspension was mixed by carefully inverting the tubes and then incubated at room temperature for 10 min. The reaction was stopped by adding 15 mL of phosphate buffered saline (PBS) (Gibco, MA, USA). Leukocytes were harvested by 10 min centrifugation at 300 g and 4 °C, and a new RBC lysis cycle was performed to lyse residual red blood cells. Leukocytes were then suspended in 0.5 mL of PBS and cells viability determined through flow cytometry (EC800, Sony Biotechnologies Inc, CA, USA), using propidium iodide staining (5µg /mL, Sigma, MO, USA). This experiment was performed three independent times, using blood from different donors. Representative flow cytometry plots are presented in Fig. S1.