The mDCT cells were cultured on a 96-black, clear bottom tissue culture plate (Corning), after transfection (24 h). The Mg2+ imaging of transfected cells was analyzed with Magnesium Green™ (Molecular Probes) as described previously (Yamazaki et al., 2013 (link); Chen et al., 2018 (link)), with slight modifications. The cells were incubated with Mg2+ loading buffer including 2 μM Magnesium Green (Molecular Probes) at 37°C for 60 min. Then, the buffer was changed to buffer without Mg2+ (MgCl2 was replaced with 60 mM NaCl) and the fluorescent was recorded at 1-min intervals. The cells images were detected by ImageXpress Micro XLS (molecular devices) and fluorescence was measured using MetaXpress High content image acquisition and analysis software (Molecular Devices). The cell fluorescence was analyzed by the software setting for Cell Scoring.
Magnesium green
Manufactured by Thermo Fisher Scientific
Sourced in China, Germany, United States, United Kingdom
Magnesium Green is a fluorescent dye used to detect and quantify magnesium ions in biological samples. It binds to magnesium, producing a fluorescent signal that can be measured using a fluorometer or fluorescence microscope. The intensity of the fluorescent signal is proportional to the concentration of magnesium ions present in the sample.
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Lab products found in correlation
Calcein am, by Merck Group (2 mentions)
Cocl2, by Merck Group (2 mentions)
Imagexpress micro xl, by Molecular Devices (1 mentions)
Metaxpress high content image acquisition and analysis software, by Molecular Devices (1 mentions)
Atp determination kit, by Thermo Fisher Scientific (1 mentions)
Bongkrekic acid, by Cayman Chemical (1 mentions)
Cck 8, by Dojindo Laboratories (1 mentions)
Ab110322, by Abcam (1 mentions)
Ab34726, by Abcam (1 mentions)
Pluronic acid, by Thermo Fisher Scientific (1 mentions)
Ogb 1, by Thermo Fisher Scientific (1 mentions)
ω agatoxin iva, by Alomone (1 mentions)
ω conotoxin gvia, by Alomone (1 mentions)
Dnase 1, by Takara Bio (1 mentions)
Anti β actin mab, by Merck Group (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Random primer, by Takara Bio (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Digitonin, by Merck Group (1 mentions)
Calcium green 5n, by Thermo Fisher Scientific (1 mentions)
11 protocols using magnesium green
1
Magnesium Imaging of Transfected Cells
2
Intracellular Magnesium Measurement in Parasites
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Freshly lysed parasites were harvested and centrifuged at 1,000 × g for 10 min. The parasites were resuspended with 1 mL of prewarmed buffer A (116 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO4, 5.5 mM d -glucose, and 50 mM HEPES [pH 7.4]) with 5 mM Magnesium Green (Invitrogen) and incubated at 37°C for 30 min. After incubation, the parasites were washed with buffer A two times to remove extracellular Magnesium Green and then resuspended with buffer A at a concentration of 3 × 107 parasites/mL and incubated at 37°C for another 30 min to allow the complete deesterification of intracellular AM esters. After incubation, 100 μL of parasites was loaded into each well of a 96-well black/clear-bottom plate. A Synergy Plate Reader was used to measure fluorescence with excitation at 488 nm and emission at 531 nm.
3
Mitochondrial Apoptosis Regulation Protocol
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Reagents were purchased as follows: bongkrekic acid (19079, Cayman, USA), carboxyatractyloside (19079, MCE, China), cisplatin (HY‐17394, MCE, China), calcein‐AM (17783, Sigma‐Aldrich, USA), CoCl2 (V900021, Sigma‐Aldrich, USA), calcimycin (A23187, HY‐N6687, MCE, China), Magnesium green™ (M3733, Invitrogen, USA), STMRM, (T668, Invitrogen, USA), ATP Determination Kit (A22066, Invitrogen, USA), CCK‐8 (CK04, Dojindo, Japan), GST tag Purification Kit (P2262, Beyotime, China), MultiS Fast Mutagenesis Kit (C214, Vazym, China), 3,5‐difluoro‐L‐tyrosine (CAS 73246‐30‐7, Yiji Biotechnology, Shanghai, China).
Antibodies used included ANT1 mAb (ab110322, Abcam, UK, 1:1,000), VDAC1 mAb (Ab34726, Abcam, UK, 1:1,000), HSP60 mAb (Sc‐1052, Santa Cruz Biotechnology, USA, 1:1,000), β‐actin mAb (A5441, Sigma, Germany, 1:10,000), cyto C mAb (556433, BD, USA, 1:1,000), TIM23 mAb (11123‐1‐AP, Proteintech, USA, 1:1,000), ANT2 mAb (14671S, CST, USA, 1:2,000), VDAC2 mAb (11663‐1‐AP, Proteintech, USA, 1:1,000), caspase‐9 mAb (9502S, CST, USA, 1:1,000), Flag mAb (F1804, Sigma‐Aldrich, USA, 1:1,000), and cleaved caspase‐3 mAb (9664S, CST, USA, 1:1,000).
Antibodies used included ANT1 mAb (ab110322, Abcam, UK, 1:1,000), VDAC1 mAb (Ab34726, Abcam, UK, 1:1,000), HSP60 mAb (Sc‐1052, Santa Cruz Biotechnology, USA, 1:1,000), β‐actin mAb (A5441, Sigma, Germany, 1:10,000), cyto C mAb (556433, BD, USA, 1:1,000), TIM23 mAb (11123‐1‐AP, Proteintech, USA, 1:1,000), ANT2 mAb (14671S, CST, USA, 1:2,000), VDAC2 mAb (11663‐1‐AP, Proteintech, USA, 1:1,000), caspase‐9 mAb (9502S, CST, USA, 1:1,000), Flag mAb (F1804, Sigma‐Aldrich, USA, 1:1,000), and cleaved caspase‐3 mAb (9664S, CST, USA, 1:1,000).
4
ADP/ATP Exchange Rate Measurement in Myotubes
5
Preparation of Chemical Stock Solutions
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All chemicals were obtained from Sigma (Taufkirchen, Germany) or Carl Roth (Karlsruhe, Germany). OGB-1, Magnesium Green, and pluronic acid were obtained from Invitrogen (LifeTechnologies GmbH, Darmstadt, Germany). Tetrodotoxin (TTX), TTA-P2, ω-conotoxin GVIA, and ω-agatoxin IVa were obtained from Alomone Labs (Jerusalem, Israel). Stock solutions were prepared according to manufacturer’s instructions and stored at −20°C.
6
ADP/ATP Exchange Rate Measurement in Myotubes
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ADP/ATP exchange rate by magnesium green (Invitrogen) was determined in permeabilized myotubes as described previously 42 (link). Briefly, C2C12 myoblasts were transfected with WT, K33Q, K43E, A90D, V289M, A114P ANT1 adenovirus. C2C12 myoblasts were induced differentiation into myotubes by replacing culture medium with differentiation medium containing DMEM and 2% house serum. Myotubes were processed on the 6th day for subsequent experiments. Cells were washed once and resuspended in a buffer containing 8 mM KCl, 110 mM K-gluconate, 10 mM NaCl,10 mM Hepes, 10 mM KH2PO4, 0.005 mM EGTA, 10 mM mannitol, 0.5 mM MgCl2. Digitonin and MgG 5 K+ salt were subsequently added. MgG fluorescence was recorded at a 0.33-Hz acquisition rate by using 505- and 535-nm excitation and emission wavelengths, respectively. Mitochondrial phosphorylation was started by the addition of 2 mM ADP. At the end of each experiment, minimum fluorescence was measured after the addition of 5 mM EDTA, and maximum fluorescence was measured by the addition of 10 mM MgCl2. ADP-ATP exchange rates were calculated by linear regression of the magnesium green fluorescence calibrated and converted to ATP appearing in the medium42 (link).
7
Intracellular Calcium Measurement in Parasites
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Freshly lysed parasites were harvested centrifugated at 1000 ×g for 10 minutes. The parasites were resuspended with 1 ml of pre-warmed Buffer A (116 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO4, 5.5 mM D-glucose, and 50 mM Hepes, pH 7.4) with 5mM Magnesium Green (Invitrogen), and incubated at 37 ˚C for 30 min. After incubation, the parasites were washed with Buffer A two times to remove extracellular Magnesium Green, then resuspended with Buffer A at a concentration of 3x10 7 parasites/ml and incubated at 37 ˚C for another 30 min to allow the complete de-esterification of intracellular AM esters. After incubation, 100 µl of parasites were loaded to each well of a 96 Well Black/Clear Bottom Plate. A Synergy Plate Reader was used to measure fluorescence with Excitation 488 nm and Emission 531 nm.
8
Mitochondrial function analysis protocol
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The materials used were as follows: lipofectamine™ 2000 transfection reagent (Life Technologies, Grand Island, NY, USA), lentivirus vector pWPXL (from Addgene, Cambridge, MA, USA), protease inhibitors (Cat#: 04693116001; Roche Molecular Biochemicals, Indianapolis, IN, USA), 9E10 mAb (anti‐c‐myc; ab32; Abcam, USA), M2 mAb (anti‐flag; F1804; Sigma‐Aldrich), anti‐ANT1 mAb (ab110322; Abcam, Cambridge, MA, USA), anti‐SOD2 mAb (12656‐RP02; Sino Biological, Beijing, China), BCL‐2 (BS1511; Bioworld Technology, Nanjing, China), Cyt c (AC909; Beyotime, Guangzhou China), COX IV (4850; Cell Signaling Technology, Beverly, MA, USA), anti‐β‐actin mAb (AC‐15; Sigma‐Aldrich), TaqMan Copy Number Reference Assay (4403316; Life Technologies), FK506 (F4679; Sigma‐Aldrich), actinomycin D (A9415; Sigma‐Aldrich), DNAseI (2270A; Takara), CHX (S1560; Beyotime), digitonin (D141; Sigma‐Aldrich), random primer (3801; Takara), Magnesium Green (M‐3733; Life Technologies); carboxyatractyloside (C4992; Sigma‐Aldrich), calcein‐AM (17783; Sigma‐Aldrich), CoCl2 (V900021; Sigma‐Aldrich), ionomycin (S1672; Beyotime), Bongkrekic acid (1820‐100; Biovision, Milpitas, CA, USA), Calcium green‐5N (c‐3737; Life Technologies), and DAPI (D9542; Sigma‐Aldrich); Roche‐In Situ Cell Death Detection Kit (12156792910; Roche Molecular Biochemicals).
9
Measuring Intracellular Magnesium in Yeast
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Exponentially growing yeast cells were harvested (5.0 × 107) in 0.75 ml of growth media and 0.25 ml of growth media containing 1 mM MagnesiumGreen (Life Technologies: M-3735) was added. Cell-MagnesiumGreen mixture was allowed to incubate for 45 min at 30°C with rotation. Cells were washed three times in growth media and allowed to incubate for an additional 30 min at 30°C with rotation. A total of 0.25 ml of cells was transferred to clear flat-bottomed 96-well plate. Absorbance was measured with a Perkin Elmer Victor3 plate reader. Relative magnesium concentration in arbitrary absorbance units was calculated using Beer's law and the following formula: [CellsMg2+] / [MediaMg2+] = AbsorbanceCells / AbsorbanceMedia, where ‘Media’ represents the growth media/magnesium-green mixture.
10
Magnesium-Dependent ADP Binding Assay
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Magnesium green was purchased from Thermo Fisher Scientific (Renfrew, UK), ADP was purchase from Merck (Hoddesdon, UK), water was purchased from VWR (Lutterworth, UK). All other chemicals were purchased from Sigma‐Aldrich (Dorset, UK).
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