Flow Cytometry-based Measurement of Reactive Oxygen Species in Cyanobacteria

Cyanobacteria are Gram-negative oxygen-producing photosynthetic bacteria that are useful in the pharmaceutical and biofuel industries. Monitoring of oxidative stress under fluctuating environmental conditions is important for determining the fitness, survival, and growth of cyanobacteria in the laboratory as well as in large scale cultivation systems. Here, we provide a protocol developed using unicellular Synechococcus elongatus PCC 7942 and filamentous Fremyella diplosiphon BK14 cyanobacteria for high-throughput oxidative stress measurement by 2',7'-dichlorodihydrofluorescein-diacetate (DCFH-DA) and flow cytometry (FCM). We also provide details for the optimization of cell number, dye concentration, and FCM parameters for each organism before it can be utilized to quantify reactive oxygen species (ROS). FCM-based method can be used to measure ROS in a large population of cyanobacterial cells in a high-throughput manner. Graphical abstract.