A Spectrofluorophotometrical Method Based on Fura-2-AM Probe to Determine Cytosolic Ca2+ Level in Pseudomonas syringae Complex Bacterial Cells

Simone Trabalza; Roberto Buonaurio; Alberto M Del Pino; Carlo A Palmerini; Harrold A van den Burg; Chiaraluce Moretti

doi:10.21769/BioProtoc.3949

A Spectrofluorophotometrical Method Based on Fura-2-AM Probe to Determine Cytosolic Ca²⁺ Level in Pseudomonas syringae Complex Bacterial Cells

Bio Protoc. 2021 Mar 20;11(6):e3949. doi: 10.21769/BioProtoc.3949.

Authors

Simone Trabalza^{1

2}, Roberto Buonaurio¹, Alberto M Del Pino¹, Carlo A Palmerini¹, Harrold A van den Burg², Chiaraluce Moretti¹

Affiliations

¹ Department of Agricultural, Food and Environmental Science, University of Perugia, Perugia, Italy.
² Molecular Plant Pathology, Swammerdam Institute for Life Sciences (SILS), University of Amsterdam, Amsterdam, the Netherlands.

Abstract

Calcium signaling is an emerging mechanism by which bacteria respond to environmental cues. To measure the intracellular free-calcium concentration in bacterial cells, [Ca²⁺]_i, a simple spectrofluorometric method based on the chemical probe Fura 2-acetoxy methyl ester (Fura 2-AM) is here presented using Pseudomonad bacterial cells. This is an alternative and quantitative method that can be completed in a short period of time with low costs, and it does not require the induction of heterologously expressed protein-based probes like Aequorin. Furthermore, it is possible to verify the properties of membrane channels involved in Ca²⁺ entry from the extracellular matrix. This method is in particular valuable for measuring [Ca²⁺]_i in the range of 0.1-39.8 µM in small cells like those of prokaryotes.

Keywords: Cytosolic calcium concentration; Fura 2-AM; Live cell signaling; Pseudomonad; Spectrophotometer.