The contents of phosphate (PO4−, #BML-AK111, BIOMOL® Green, Enzo, Farmingdale, NY, USA), calcium (Ca+2, #MAK022, Sigma-Aldrich), and chloride (Cl−, #MAK023, Sigma-Aldrich) from the plasma were quantified using colorimetric assays following the manufacturer’s instructions. For the phosphate, the linear range of detection was between 0.03–2 nmol/well. For the calcium, the linear range of detection was between 0.4–2.0 μg/well. For the chloride, the linear range of detection was between 20–100 nmol/well. All the analytes were first evaluated and validated in the plasma from other teleost fish [28 (link),29 (link),30 ].
Mak022
Manufactured by Merck Group
Sourced in United States, France
MAK022 is a laboratory instrument designed for general purpose analysis. It features high-precision measurement capabilities and is suitable for a variety of applications in research and testing environments.
Automatically generated - may contain errors
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26 protocols using mak022
1
Colorimetric Plasma Analyte Quantification
2
Serum Calcium Ion Quantification
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When the mice were sacrificed, blood was collected from the inferior vena cava and stored in single-use plastic tubes on ice for 15 minutes. Serum was separated by centrifugation at 1100 g for 10 minutes at 4°C and stored at −80°C for the subsequent analysis. Calcium ion content in the serum was determined using a commercially available colorimetric assay kit (MAK022, Sigma-Aldrich, St. Louis, MO, USA) in accordance with the manufacture’s guidelines. Calcium ion content in the samples were determined using a standard curve generated by measuring a series of samples with known concentrations of ionized calcium. All samples were analyzed in duplicates.
3
Serum Calcium Determination Protocol
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The blood was collected by inferior palpebral vein puncture, kept for 2 h at 4 °C, and centrifuged at 400× g for 10 min at 4 °C. The serum calcium levels were determined using colorimetric assays (MAK022, Sigma Aldrich, Saint-Quentin-Fallavier, France), according to the supplier’s protocol.
4
Quantifying VIC Calcification with Cresolphthalein
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VIC calcification was assessed using the o-cresolphthalein method (MAK022, Merck Sigma). Calcium deposits were collected as 0.6 N HCl extracts, as previously published [35 (link), 36 (link)]. Cell monolayers were homogenized in 0.1% SDS, 0.1 M NaOH, 5 mM EDTA buffer for ulterior normalization of the calcification readouts. VICs were assayed in technical quadruplicates.
5
Colorimetric calcium quantification
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Supernatants from each timepoint in the ex vivo culture were retrieved and analysed for calcium concentration using a colorimetric assay (Sigma-Aldrich MAK022; n = 3 for each condition). The chromogenic reagent (o-Cresolphthalein complexone) forms a violet-coloured complex (absorbance: 575 nm) with Calcium ions (Ca2+) in alkaline solution. Absorbance readings were taken in triplicate for each sample, fresh media, and blank media without any reagent, as per manufacturer’s instructions.
6
Quantifying In Vitro Calcification in VICs
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In vitro calcification was assessed in VICs after 9 days in culture in medium supplemented with 2.6‐mM phosphate. Calcification was quantified by extracting the calcium‐phosphate salts with 0.6‐N HCl and quantified using a calcium colorimetric assay (Sigma‐Aldrich, MAK022) according to manufacturer's protocol.
7
Quantifying Calcium in FBS
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Calcium ion content in FBS was quantified via a colorimetric assay (#MAK022, Sigma-Aldrich) following manufacturer’s instructions. Results are represented as mean ± SD in three replicates.
8
Measuring Glucose and Calcium Levels
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The supernatants were collected and frozen at −80 °C until used. Glucose and calcium concentrations in the medium were measured using enzymatic assay kits EIAGLUC from ThermoFisher Scientific for glucose, and MAK022 from Sigma for calcium detection. The assays were conducted following the manufacturer's instructions.
9
Calcium Release from Polymer Membranes
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Calcium release from particles was studied in duplicates by immersing 1 cm2 of the membranes from PEU + 45% Ca-TMP and pure PEU groups (n = 3) in 1 mL of PBS and lipase at 37 °C. During the 21-day period, we used the supernatant to measure calcium levels using the calcium colorimetric assay kit (MAK022, Sigma Aldrich) according to the manufacturer’s instructions. This kit detects the chromogenic complex formed between calcium ions and o-Cresolphthalein to determine the concentration of calcium ions. The absorbance, which is directly proportional to the concentration of calcium ions present in the sample of this complex, was read at 575 nm on a plate reader (Spectra Max Id3, Molecular Devices LLC, San Jose, CA, USA), and it was calculated from a standard calcium solution prepared in parallel. The values obtained by reading the PEU group were used as background and subtracted from the PEU + 45% Ca-TMP group to obtain the actual reading.
10
Calcium Content Determination Protocol
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For the calcium analysis, samples were lyophilized, weighed, and hydrolyzed with 6M hydrochloric acid. Calcium content was determined using a colorimetric assay (Sigma MAK022).
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