Synthesis of a highly Mg2+-selective fluorescent probe and its application to quantifying and imaging total intracellular magnesium.
Magnesium plays a crucial role in many physiological functions and pathological states. Therefore, the evolution of specific and sensitive tools capable of detecting and quantifying this element in cells is a very desirable goal in biological and biomedical research. We developed a Mg2+-selective fluorescent dye that can be used to selectively detect and quantify the total magnesium pool in a number of cells that is two orders of magnitude smaller than that required by flame atomic absorption spectroscopy (F-AAS), the reference analytical method for the assessment of cellular total metal content. This protocol reports itemized steps for the synthesis of the fluorescent dye based on diaza-18-crown-6-hydroxyquinoline (DCHQ5). We also describe its application in the quantification of total intracellular magnesium in mammalian cells and the detection of this ion in vivo by confocal microscopy. The use of in vivo confocal microscopy enables the quantification of magnesium in different cellular compartments. As an example of the sensitivity of DCHQ5, we studied the involvement of Mg2+in multidrug resistance in human colon adenocarcinoma cells sensitive (LoVo-S) and resistant (LoVo-R) to doxorubicin, and found that the concentration was higher in LoVo-R cells. The time frame for DCHQ5 synthesis is 1-2 d, whereas the use of this dye for total intracellular magnesium quantification takes 2.5 h and for ion bioimaging it takes 1-2 h.
Department of Pharmacy and Biotechnologies, University of Bologna, Bologna, Italy.
Azzurra
A
Sargenti
Department of Pharmacy and Biotechnologies, University of Bologna, Bologna, Italy.|National Institute of Biostructures and Biosystems, Rome, Italy.
Giovanna
G
Farruggia
Department of Chemistry 'Giacomo Ciamician', University of Bologna, Bologna, Italy.
Nelsi
N
Zaccheroni
ASMN-IRCCS, Department of Transfusion Medicine, Reggio Emilia, Italy.
Chiara
C
Marraccini
Department of Chemistry 'Giacomo Ciamician', University of Bologna, Bologna, Italy.
Massimo
M
Sgarzi
Department of Pharmacy and Biotechnologies, University of Bologna, Bologna, Italy.
Concettina
C
Cappadone
Department of Pharmacy and Biotechnologies, University of Bologna, Bologna, Italy.
Emil
E
Malucelli
Department of Pharmacy and Biotechnologies, University of Bologna, Bologna, Italy.
Alessandra
A
Procopio
Department of Chemistry 'Giacomo Ciamician', University of Bologna, Bologna, Italy.
Luca
L
Prodi
Department of Chemistry 'Giacomo Ciamician', University of Bologna, Bologna, Italy.
Marco
0000-0001-8415-8363
M
Lombardo
Department of Pharmacy and Biotechnologies, University of Bologna, Bologna, Italy.|National Institute of Biostructures and Biosystems, Rome, Italy.
Stefano
S
Iotti
D045744:Cell Line, Tumor; D060326:Chemistry Techniques, Synthetic / Q000379:methods*; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006912:Hydroxyquinolines / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D042541:Intracellular Space / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism*; D008856:Microscopy, Fluorescence
D005456:Fluorescent Dyes; D006912:Hydroxyquinolines; D008274:Magnesium