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. 2021 Mar 20;11(6):e3949.
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

Simone Trabalza  1   2 , Roberto Buonaurio  1 , Alberto M Del Pino  1 , Carlo A Palmerini  1 , Harrold A van den Burg  2 , Chiaraluce Moretti  1
Affiliations

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

Simone Trabalza et al. Bio Protoc. .

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.

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Conflict of interest statement

Competing interestsThe authors declare no conflict-of-interest and have no competing financial interests. Informed consent was obtained from all individual participants included in the study.

Figures

Figure 1.
Figure 1.. The fate of Fura 2-AM in cells.
Fura 2-AM is de-esterified by cellular esterases and transformed in Fura 2, which is able to form a complex with cytosolic calcium (Ca2+) and cannot passively cross the cell membrane.
Figure 2.
Figure 2.. Excitation spectra of Fura 2.
Excitation spectra of Fura 2 in solution containing 0 to 39.8 µM of free calcium (Ca2+). Modified from Grynkiewicz et al. (1985) .
Figure 3.
Figure 3.. FL WinLab Software.
A. The Traffic light button allows to start and stop the analysis and describes the status of the instrument. B. The Calibration Tab layout permits to manage the raw data and the Convert to [ion] button to visualize the data (from the FL WinLab User’s Guide, PerkinElmer, Inc., UK).
Figure 4.
Figure 4.. Example of a plot generated by FL WinLab Software used to calculate Δ[Ca2+]c before and after CaCl2 addition.
The presence of the peaks in the trace is due to the change of the wavelength that LS-50B Luminescence Spectrometer shifts every two seconds. Therefore, the calculation of the Ca2+ concentration has to be performed considering the baseline (dotted line). After about 50 s, when the signal is stabilized, the CaCl2 is added (red arrow) and the Δ[Ca2+]c is calculated between the highest Ca2+ concentration value reached in the baseline (blue arrow) and the value when the CaCl2 is added (red arrow).
Figure 5.
Figure 5.. An increase of cytosolic Ca2+ levels in Pseudomonas savastanoi pv. savastanoi (Psav) DAPP-PG 722.
Psav bacterial cells incubated in HBSS medium alone (basal condition, closed squares) or in the presence of glucose, fructose, sucrose, indole 3 acetic acid (IAA) or tryptophan (open circles) over a concentration range of extracellular calcium chloride. Each point represents the mean of 10 independent experiments ± SE. From Moretti et al. (2019) .
See this image and copyright information in PMC

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