From mirage to reality: an improved Mg assay to assess Magnesium modulation in young and senescent Bone Marrow Mesenchymal Stem Cells

Purpose: Magnesium is reported to modulate the differentiation of bone marrow mesenchimal stem cells (BMSc). At the moment no data are available about Mg homeostasis in these cell. We compared BMSc at various population doublings (PD) for their intracellular content of total Mg in parallel with the expression of some of its channels and transporters. Given the low number of senescent cells available, it was necessary to develop a specific protocol to miniaturize the assay with the fluorescent probe DCHQ5. Material and method: BMSc were harvested, extensively washed, counted and pelleted. Intracellular total Mg was assessed on samples with at least 3000 cells. The pellet was resuspended in MOPS 4mM, in a volume of at least 200 uL, sonicated at 100W, and diluted in methanol. DCHQ5 was added to a final concentration of 15 uM. The fluorescence was read with λex 362 nm and λem 510 nm using a 96 black-well plate in a fluorescence plate reader. The quantification was performed by using a calibration curve with MgSO4 diluted in MOPS:MetOH 1:1. The expression of TRPM7, MAGT1, SLC41A1, CNNM2 in young and senescent cells was evaluated by RT-PCR. Confocal microscopy was performed to detect the mitochondria. Results: The miniaturized DCHQ5 assay revealed the gradual decrease of intracellular Mg with the increase of PD. In parallel, a reduced expression of TRPM7 and MagT1 was detected, whereas SCL41a1 and CNNM2 were not modulated. Since Mg is localized in the mitochondria, it is of note that senescent cells possess less mitochondria, which are much more fragmented that in young cells. Conclusion: The novel DCHQ5 miniaturized assay is a useful tool to study intracellular Mg homeostasis in very small samples. BMSc senescence is associated with the decrease of intracellular Mg, the downregulation of TRPM7 and MagT1, and reduced number of mitochondria.