Abstract
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.
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Acknowledgements
This work was supported by the RFO (Ricerca Fondamentale Orientata) of the University of Bologna.
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A.S., G.F., C.C., M.L., N.Z., E.M., A.P., C.M. and M.S. conducted the experiments; M.L., S.I., L.P. and G.F. designed the experiments; and A.S., M.L., G.F. N.Z. and S.I. wrote the paper.
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Integrated supplementary information
Supplementary Figure 4 Purification of of 5-phenyl-8-tosyloxyquinoline (3).
TLC (cyclohexane:ethyl acetate 75:25) of flash-chromatography relative to the purification of the Suzuki cross-coupling reaction for the synthesis of 5-phenyl-8-tosyloxyquinoline 3; R = real purified product (3); 6-16 = eluted fractions. UV lamp, 254 nm.
Supplementary Figure 8 TLC check of the Suzuki cross-coupling reaction.
TLC (cyclohexane:ethyl acetate 75:25) of Suzuki cross-coupling for the synthesis of 5-phenyl-8-tosyloxyquinoline 3. S = starting material (5-chloro-8-tosyloxyquinoline, 2); P = crude reaction mixture; R = real purified product (3). UV lamp, 254 nm.
Supplementary Figure 9 TLC check of the hydrolysis reaction.
TLC (cyclohexane:ethyl acetate 75:25) of basic hydrolysis deprotection for the synthesis of 5-phenyl-8-hydroxyquinoline 4. S = starting material (5-phenyl-8-tosyloxyquinoline, 3); PE = crude extracted reaction mixture; R = real purified product (4). UV lamp, 254 nm.
Supplementary Figure 13 TLC check of the Mannich reaction.
TLC check (dichloromethane:methanol 9:1 + 2.5% aqueous NH4OH) of the Mannich reaction for the synthesis of DCHQ5. A = 1,4,10,13-tetraoxa-7,16-diazacyclooctadecane (5); S = starting material (5-phenyl-8-hydroxyquinoline, 4); P = crude reaction mixture; R = real purified product (DCHQ5). Left) UV lamp, 254 nm, right) UV lamp, 310 nm.
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Supplementary Table 1 and Supplementary Figures 1–16. (PDF 1108 kb)
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Sargenti, A., Farruggia, G., Zaccheroni, N. et al. Synthesis of a highly Mg2+-selective fluorescent probe and its application to quantifying and imaging total intracellular magnesium. Nat Protoc 12, 461–471 (2017). https://doi.org/10.1038/nprot.2016.183
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DOI: https://doi.org/10.1038/nprot.2016.183
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