HOS cell-containing collagen solution (100 µL) was plated in each well of 96-well plates, and the culture plates were incubated for 30 min at 37 °C for gelation before addition to the culture medium. The cell-containing gel turbidity was measured at days 1, 2, 3, 4, 5, 6, and 7, then the gel samples were collected daily and centrifuged at 13, 000 × g for 10 min at 4 °C, after which the supernatant was removed. The precipitate was treated with formic acid (30 µL) and incubated at 4 °C for 24 h. Then, the dissolved calcium contents of the supernatant were measured via the Calcium E-test (Wako) according to the manufacturer’s instructions.
Calcium e test
Manufactured by Fujifilm
Sourced in Japan
The Calcium-E test is a laboratory equipment product designed to measure the calcium levels in biological samples. It employs a colorimetric method to determine the concentration of calcium ions in the tested solution. The core function of this product is to provide accurate and reliable calcium concentration data for various clinical and research applications.
Automatically generated - may contain errors
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12 protocols using calcium e test
1
Quantifying Calcium Content in Cell-Laden Gels
2
Quantifying Aortic Calcium Deposition
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To quantify calcium content, the lower parts of the abdominal aortas were removed. The aortas were freeze-dried, weighed, and then incubated in 0.6 N HCl for 48 h. The samples were centrifuged and the supernatant were analyzed for calcium contents using the Calcium-E test (Wako, Osaka, Japan). Aortic calcium content was corrected for the dry tissue weight. The thoracic aortas were removed and fixed in 10% formaldehyde for histopathology. These sections were dehydrated with 70% ethanol at room temperature and embedded in paraffin blocks, which were deparaffinized and processed for von Kossa staining. The percentage of the positive areas of von Kossa staining in the aortic sections were calculated in 30 randomly selected microscopic fields using Lumina Vision image analysis software version 3.7.4.2 (Mitani Co., Tokyo, Japan). All evaluations were conducted in a blinded manner.
3
Plasma Biochemical Analysis Protocol
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Plasma calcium levels were measured using the calcium-E test (Wako Chemical) according to the manufacturer’s instructions. Plasma sample (2.5 μL) was mixed with substrate buffer (100 μL) and coloring reagent (50 μL). The absorbance of the reaction mixture was measured at 610 nm.
Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured using the Transaminase CII Test Wako (Wako Chemical) according to the manufacturer’s instructions and as previously described [20 (link), 21 (link)]. Concentrations of plasma creatinine and blood urea nitrogen (BUN) were measured using Creatinine Liquid Reagents Assay (DIAZYME, Poway, CA) and BUN Wako Test (Wako Chemical), respectively, according to the manufacturer’s instructions and as previously described [22 (link), 23 (link)]. For relative quantification, calibration curves were prepared using standard solutions.
Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured using the Transaminase CII Test Wako (Wako Chemical) according to the manufacturer’s instructions and as previously described [20 (link), 21 (link)]. Concentrations of plasma creatinine and blood urea nitrogen (BUN) were measured using Creatinine Liquid Reagents Assay (DIAZYME, Poway, CA) and BUN Wako Test (Wako Chemical), respectively, according to the manufacturer’s instructions and as previously described [22 (link), 23 (link)]. For relative quantification, calibration curves were prepared using standard solutions.
4
Plasma, Fecal, and Urinary Mineral Analysis
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Plasma, feces, and urinary Pi and calcium and serum blood urea nitrogen (BUN) were determined by the Phospha-C test, Calcium-E test, or BUN-B test (Wako Pure Chemical Industries, Ltd., Osaka, Japan), respectively (40 (link)). To measure the fecal Pi and calcium levels, the collected feces (24 h) were dried at 110°C for 24 h, 250°C for 3 h, and at 350°C for 3 h. FGF23 and PTH concentrations were determined using an FGF23 ELISA kit (Kainos Laboratories, Inc., Tokyo, Japan) and mouse PTH 1-84 ELISA kit (Immutopics, CA, USA), respectively (40 (link)). 1,25(OH)2D levels were measured using a radioreceptor assay (SRL, Inc., Tokyo, Japan). Heparinized mixed arterial–venous blood was collected and analyzed immediately for pH, blood gases, and electrolytes using an OPTI CCA TS blood gas analyzer (Sysmex Corporation, Kobe, Japan).
5
Klotho Mutant Mice: Plasma Biochemistry
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All animals were maintained climate-controlled room (22 ± 2°C) with 12 h:12 h dark-light cycles under pathogen-free conditions and handled in accordance with the Guidelines for Animal experimentation of the Tokushima University. The protocol of all animal experiments were approved by Animal experiment committee of the Tokushima University.
Heterozygous klotho mutant (kl/+) mice were purchased from Japan CLEA, Inc (Osaka, Japan). Homozygous klotho mutant (kl/kl) mice were generated by crossing kl/+ mice and wild type (WT) mice. Mice were weaned at 3 weeks of age and given free access to water and standard mouse chow (Oriental Yeast, Osaka, Japan) under pathogen-free condition. WT and kl/kl mice were sacrificed at 3- and 6-week-old. For biochemical analysis of plasma, blood was collected with heparin by puncture of inferior vena cava. The plasma levels of Ca and Pi were determined by Calcium-E test (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and Phospha-C test (Wako), respectively.
Heterozygous klotho mutant (kl/+) mice were purchased from Japan CLEA, Inc (Osaka, Japan). Homozygous klotho mutant (kl/kl) mice were generated by crossing kl/+ mice and wild type (WT) mice. Mice were weaned at 3 weeks of age and given free access to water and standard mouse chow (Oriental Yeast, Osaka, Japan) under pathogen-free condition. WT and kl/kl mice were sacrificed at 3- and 6-week-old. For biochemical analysis of plasma, blood was collected with heparin by puncture of inferior vena cava. The plasma levels of Ca and Pi were determined by Calcium-E test (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and Phospha-C test (Wako), respectively.
6
Plasma Inorganic Biomarkers Quantification
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The concentrations of inorganic Pi, Ca and creatinine (Cre) were determined using the Phospha C-test Wako, the Calcium E-test Wako, and LabAssayTM Creatinine test kits, respectively (Wako, Osaka, Japan). Concentrations of intact FGF23 and intact PTH (1-84) in the plasma were determined using the FGF23 ELISA Kit (Kinos, Tokyo, Japan) and Mouse PTH 1-84 ELISA Kit (Immutopics, San Clemente, CA), respectively. Plasma 1,25(OH)2D concentrations were determined by radioimmunoassay (SRL, Tokyo, Japan).
7
Femur Bone Ash Content Analysis
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The right femur was taken and weighed after removing the meat. Ash content in the femur was first treated by drying at 105°C for 48 h, then defatted by absolute ether for 3 days. Defatted bone was treated by ashing process at 550°C overnight in an electric furnace. The ash in the femur was measured with the weight method [19 ]. Ash was then added to 10 mL of 2 N HCl, and calcium and phosphorus in ash were measured with test kits (Calcium E-Test and Phospha C-Test, Wako Pure Chemical Industries, Osaka, Japan).
8
Serum Biomarkers in Mouse Models
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Blood samples were collected using cardiac puncture of anesthetized mice and were stored at − 20 °C until chemical analysis. Serum calcium, phosphate, and creatinine levels were determined using the Calcium E-TEST, the Phospha-C TEST, and the LaboAssay Creatinine test (Wako Pure Chemical Industries Ltd), respectively. Serum PTH, FGF23, and sclerostin were measured using the Mouse PTH 1-84 enzyme-linked immunosorbent assay (ELISA) kit (Quidel, previously Immutopics), Human FGF23 ELISA Kit (Kinos) [21 (link)], and Mouse/Rat SOST Quantikine ELISA Kit (R&D Systems), respectively. All kits were used according to the manufacturers’ instructions.
9
Plasma biomarker quantification protocol
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Plasma Ca levels were measured using the calcium-E test (Wako Chemical) according to the manufacturer’s instructions. Plasma samples (2.5 μL) were mixed with the substrate buffer (100 μL) and a coloring reagent (50 μL). The absorbance of the reaction mixture was measured at 610 nm.
Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured using the Transaminase CII Test Wako (Wako Chemical) according to the manufacturer’s instructions and as previously described [15 (link), 16 ]. Concentrations of plasma creatinine and blood urea nitrogen (BUN) were measured using the Creatinine Liquid Reagents Assay (DIAZYME, Poway, CA, USA) and the BUN Wako Test (Wako Chemical), according to the manufacturer’s instructions and as previously described [17] (link). For relative quantification, calibration curves were prepared using a standard solution.
Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured using the Transaminase CII Test Wako (Wako Chemical) according to the manufacturer’s instructions and as previously described [15 (link), 16 ]. Concentrations of plasma creatinine and blood urea nitrogen (BUN) were measured using the Creatinine Liquid Reagents Assay (DIAZYME, Poway, CA, USA) and the BUN Wako Test (Wako Chemical), according to the manufacturer’s instructions and as previously described [17] (link). For relative quantification, calibration curves were prepared using a standard solution.
10
Calcium Release from Cartilage Scaffolds
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Calcium (Ca) release from CS and Ap-CS was assessed. Each piece of CS and Ap-CS was immersed in 0.5 mL of phosphate-buffered saline (PBS, FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) at 37 °C for 7 days. The amount of released Ca in PBS was measured using a calcium test kit (calciumE-test; FUJIFILM Wako Pure Chemical Corp.) and spectrophotometer (U-1100; Hitachi, Ltd.) at 610 nm.
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