Calcium AS FS (DiaSys Diagnostic Systems, Holzheim, Germany) was used to quantify calcium content (normalized to the cell number per well) after cell lysis. OsteoImageTM mineralization assay (Lonza, Basel, Switzerland) applied according to the manufacturer’s instructions, allowed for hydroxyapatite visualization and quantification via the high resolution fluorescence scanner. Cells were stained with Alexa Fluor 568 phalloidin and examined using a Leica SP5X laser scanning confocal microscope (Leica, Wetzlar, Germany) for qualitative assessment. Leica Applications Suite was used to obtain images.
Calcium as fs
Manufactured by DiaSys
Sourced in Germany
Calcium AS FS is a diagnostic reagent used to measure the total calcium concentration in human serum or plasma samples. The assay is based on the reaction of calcium with o-cresolphthalein complexone, which forms a colored complex that can be measured photometrically.
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Lab products found in correlation
Osteoimage mineralization assay, by Lonza (1 mentions)
Sp5x laser scanning confocal microscope, by Leica (1 mentions)
Applications suite, by Leica (1 mentions)
Nunc carrier plate transwell system, by Thermo Fisher Scientific (1 mentions)
Magnesium xl fs, by DiaSys (1 mentions)
Konelab 20i, by Thermo Fisher Scientific (1 mentions)
5 protocols using calcium as fs
1
Quantifying Calcium and Mineralization
2
Calcium Absorption and Mineralization Assay
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Analysis of calcium absorption through cGM was performed in a NUNC® carrier plate transwell system (insert position: medium). 5 mg of cGM were added in the upper chamber to 24 h pre-seeded SaOS-2 cells (6•104 cells/Well) and gently shaken on orbital shaker (70–80 rpm) for 30 min at 37 °C and 5% CO2. Samples were incubated under static conditions for further 24 h and medium was changed to OM. Supernatants were collected in reaction tubes. Calcium absorption was measured by Calcium AS FS (Diasys, Holzheim, Germany) according to the manual. Deficits of calcium in supernatants were calculated as absorption by the cGM.
For another experiment, calcium concentration was increased up to 15% to compensate for absorbed calcium. To analyze mineralization of cells and microtissues, medium was removed and samples were carefully washed with warm PBS (37 °C). For cell lysis, 0.5 N HCl (Th. Geyer, Berlin, Germany) was added, and samples were incubated at room temperature (RT) for 24 h during shaking. Afterwards samples were transferred to reaction tubes, incubated in an ultrasonic bath for 2 h and shaken for further 12 h at RT. Samples were measured as described above and calcium content was calculated relative to DNA content per well.
For another experiment, calcium concentration was increased up to 15% to compensate for absorbed calcium. To analyze mineralization of cells and microtissues, medium was removed and samples were carefully washed with warm PBS (37 °C). For cell lysis, 0.5 N HCl (Th. Geyer, Berlin, Germany) was added, and samples were incubated at room temperature (RT) for 24 h during shaking. Afterwards samples were transferred to reaction tubes, incubated in an ultrasonic bath for 2 h and shaken for further 12 h at RT. Samples were measured as described above and calcium content was calculated relative to DNA content per well.
3
Photometric Analysis of Extracellular Calcium
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Extracellular calcium (Ca) concentration was analyzed photometrically using a diagnostic kit (Calcium AS FS, DiaSys Diagnostic Systems, Holzheim, Germany). The principle of this test lies in the reaction with arsenazo III (120 µmol/L) at neutral pH which results in a blue complex. Ten microliters of the blank/standard/tested sample were mixed with 1000 µL of reagent. After 5 min at room temperature, the absorbance was measured at 650 nm by an RX Monza biochemical analyzer (Randox Laboratories, Crumlin, UK). The results are defined in mM/L.
4
Quantitative Measurement of Calcium and Magnesium
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The concentrations of calcium and magnesium were measured by using a diagnostic reagent for quantitative in vitro determination produced by DiaSys Diagnostic Systems (Calcium AS FS and Magnesium XL FS, DiaSys Diagnostic Systems GmbH, Holzheim, Germany). Calcium levels were measured using the Arsenazo III test according to the manufacturer's recommendations. The test measuring range was within 0.04–20.00 mg/dL. Magnesium concentrations were measured by photometric test using xylidyl blue according to the manufacturer's procedures. The test measurements range was from 0.05 to 5.00 mg/dL, and the analyzer Konelab 20i® (ThermoScientific, Vantaa, Finland) was used for determinations.
5
Spectrophotometric Determination of Calcium and Phosphate
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The plasma concentrations of ionized calcium and inorganic phosphate were determined spectrophotometrically according to the manufacturer’s protocols (Calcium AS FS and Phosphate FS both from DiaSys Diagnostic Systems, Holzheim, Germany).
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