Flow Cytometry-based Measurement of Reactive Oxygen Species in Cyanobacteria

Soumila Mondal; Shailendra P Singh

doi:10.21769/BioProtoc.4417

Flow Cytometry-based Measurement of Reactive Oxygen Species in Cyanobacteria

Bio Protoc. 2022 May 20;12(10):e4417. doi: 10.21769/BioProtoc.4417.

Authors

Soumila Mondal¹, Shailendra P Singh¹

Affiliation

¹ Centre of Advanced Study in Botany, Department of Botany, Institute of Science, Banaras Hindu University, Varanasi-221005, UP, India.

Abstract

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.

Keywords: Cyanobacteria; DCFH-DA; Flow Cytometry; Fluorescence microscopy; Oxidative stress; ROS.