Large-scale analysis of pollen viability and oxidative level using h₂DCFDA-Staining Coupled with Flow Cytometry

Determining pollen viability and other physiological parameters is of critical importance for evaluating the reproductive capacity of plants, both for fundamental and applied sciences. Flow cytometry is a powerful high-performance high-throughput tool for analyzing large populations of cells that has been in restricted use in plant cell research and in pollen-related studies, it has been minimized mostly for determination of DNA content. Recently, we developed a flow cytometry-based approach for robust and rapid evaluation of pollen viability that utilizes the reactive oxygen species (ROS) fluorescent reporter dye H₂DCFDA (Luria et al., Plant J 98(5):942–952, 2019). This new approach revealed that pollen from Arabidopsis thaliana and Solanum lycopersicum naturally distribute into two subpopulations with different ROS levels. This method can be employed for a myriad of pollen-related studies, primarily in response to stimuli such as biotic or abiotic stress. In this chapter, we describe the protocol for H₂DCFDA staining coupled with flow cytometry analysis providing specific guidelines. These guidelines are broadly applicable to many other types of cellular reporters to further develop this novel approach in the field of pollen biology.