Calcium e test wako

Manufactured by Fujifilm

Sourced in Japan

The Calcium E-Test Wako is a laboratory test kit designed to measure the concentration of calcium in biological samples. It provides a quantitative analysis of calcium levels through a colorimetric method. The product is intended for use in clinical and research settings by qualified laboratory personnel.

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20 protocols using calcium e test wako

Evaluating Osteogenic Maturation of hMSC/HUVEC Constructs

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Sections (5-μm thick) of the hMSC/HUVEC constructs cultured for 50 days were prepared and von Kossa staining or Alcian blue staining were conducted. The stained areas of mineralized aggregates were measured using images from the CCD camera (DS-Fi2) equipped with a light microscope (ECLIPSE Ci; Nikon). Four sections from four different cell constructs were used in the quantitative analysis.
The hardness of the mineralized aggregate was measured for investigating osteogenic-maturation of the hMSC/HUVEC constructs. Briefly, hMSC/HUVEC constructs cultured for 50 days were embedded in paraffin and then cut to the core using a microtome. The consistencies of the fabricated tissues were evaluated for micro-Vickers hardness using a micro hardness testing machine (MicroWiZhard; Mitsutoyo, Kawasaki, Japan) (n = 5).
The amount of mineral deposition produced by hMSCs in the cell constructs was investigated using the methylxylenol blue (MXB) method (Calcium E-Test Wako; Wako). hMSC/HUVEC cell constructs (100:0–95:5) cultured for 50 days were washed with ultrapure water and dried using a dry-heat sterilizer (MOV-112S; Sanyo, Tokyo, Japan). Dried cell constructs were dissolved in 1 M HCl and mixed with MXB and monoethanolamine buffer. The calcium concentrations of the solutions were determined by measuring the absorbance at 595 nm (n = 4).

Sasaki J.I., Hashimoto M., Yamaguchi S., Itoh Y., Yoshimoto I., Matsumoto T, & Imazato S. (2015). Fabrication of Biomimetic Bone Tissue Using Mesenchymal Stem Cell-Derived Three-Dimensional Constructs Incorporating Endothelial Cells. PLoS ONE, 10(6), e0129266.

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Measurement of Plasma Calcium Levels

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The plasma Ca concentration was measured using the Calcium E-Test Wako (Wako Pure Chemical, Osaka, Japan)^{6 (link)}.

Kise S., Iijima A., Nagao C., Okada T., Nishikawa M., Ikushiro S., Nakanishi T., Sato S., Yasuda K, & Sakaki T. (2023). Gene therapy for alopecia in type II rickets model rats using vitamin D receptor-expressing adenovirus vector. Scientific Reports, 13, 18528.

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Osteogenic Differentiation of ASCs

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ASCs (passage 5) were seeded in triplicate in a 24-well plate (Falcon^®) at a density of 4 × 10⁴ in D/α medium. After 4 days, differentiation was carried out using osteogenic differentiation medium consisting of DMEM (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Sigma-Aldrich), 50 μM ascorbic acid 2-phosphate (Sigma-Aldrich), 0.01 μM Deamethasone (Sigma-Aldrich) and 1% Antibiotic-Antimycotic (Gibco) for 21 days. Culture medium was replaced every 2–3 days. After induction was completed, the cell densities were determined using cck8.
Osteogenesis was assayed by calcium content in differentiated cells and matrix mineralization with Alizarin Red S staining. The total calcium content was determined by a colorimetric assay using Calcium E-Test Wako (Wako Pure Chemical Industries, Osaka, Japan) after eluted by 10% (w/v) Formic acid (Wako) [31] (link). By the same method as for adipogenesis, chondrogenic potential was evaluated by the values adjusted by standard cells.

Kawagishi-Hotta M., Hasegawa S., Igarashi T., Yamada T., Takahashi M., Numata S., Kobayashi T., Iwata Y., Arima M., Yamamoto N., Yagami A., Nakata S., Uzawa T., Matsunaga K., Sugiura K, & Akamatsu H. (2017). Enhancement of individual differences in proliferation and differentiation potentials of aged human adipose-derived stem cells. Regenerative Therapy, 6, 29-40.

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Spectrophotometric Calcium Quantification

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Three pellets (5 μL each) were mixed with 2.5 μL of 0.5 N HCl in a tube and left to stand for 10 min. Samples were centrifuged at 13,000 rpm for 5 min at 4 °C and the supernatant (2.5 μL) was transferred into a 96-well plate. The calcium concentration in the supernatant was spectrophotometrically determined at 595 nm by following the color development with a calcium assay kit (Calcium E-test Wako, Fujifilm Wako Pure Chemical Industries, Ltd., Osaka, Japan). All values were normalized against the cultivation area.

Nonoyama S., Karakida T., Chiba-Ohkuma R., Yamamoto R., Ujiie Y., Nagano T., Yamakoshi Y, & Gomi K. (2021). Development and Characterization of Alkaline Phosphatase-Positive Human Umbilical Cord Perivascular Cells. Cells, 10(11), 3011.

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Serum Biomarkers of Bone Metabolism

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At 6 weeks of age, the blood was collected at 12:00 (noon). Bone Specific Alkaline Phosphatase (BAP) and tartrate-resistant acid phosphatase 5b (TRACP-5b) were measured using a specific enzyme immunoassay (CUSABIO BIOTECH, Ltd, Wuhan, China). The receptor activator of NF-κB ligand (RANKL) was measured using ELISA kit (R&D SYSTEMS, Minneapolis, MN, USA). Osteocalcin was measured using a MOUSE OSTEOCALCIN EIA KIT (Biomedical Tec Inc., Stoughton, MA, USA). Serum Ca level was measured using a calcium E-test WAKO (WAKO, Tokyo, Japan). β-estradiol (E2) was measured using an Estradiol EIA Kit (Cayman Chemical Co., Ann Arbor, MI, USA). IGF-1 was measured using an Assay Max Mouse IGF-1 ELISA kit (ASSAYPRO, St. Charles, MO, USA). Growth hormone (GH) was measured using a Rat/Mouse Growth Hormone ELISA kit (MILLIPORE, Billerica, MA, USA). Insulin was measured using a sensitive mouse insulin ELISA kit (Shibayagi, Gumma, Japan).

Sasanuma H., Nakata M., Parmila K., Nakae J, & Yada T. (2017). PDK1-FoxO1 pathway in AgRP neurons of arcuate nucleus promotes bone formation via GHRH-GH-IGF1 axis. Molecular Metabolism, 6(5), 428-439.

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Serum Calcium and 1,25(OH)2D3 Quantification

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The serum concentrations of total calcium (mg/dL) and 1,25(OH)₂D₃ (pg/mL) were assayed using the Calcium E-Test Wako (Fujifilm Wako Pure Chemical Co., Osaka, Japan) and 1,25(OH)₂ D RIA kit FR (Fujirebio Inc., Tokyo, Japan), respectively, according to the manufacturer’s instructions.

Kuwata N., Mukohda H., Uchida H., Takamatsu R., Binici M.M., Yamada T, & Sugiyama T. (2024). Renal Endocytic Regulation of Vitamin D Metabolism during Maturation and Aging in Laying Hens. Animals : an Open Access Journal from MDPI, 14(3), 502.

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Serum Biomarker Measurement Protocol

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Serum markers were measured using the following assay reagents: serum intact PTH, rat intact PTH ELISA kit (cat. no. 60-2500, Immutopics, Inc., San Clemente, CA, USA, RRID: AB_2827505); serum Ca, Calcium E-Test Wako (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan); serum Pi, Phospha C-Test Wako (FUJIFILM Wako Pure Chemical Corporation); serum FGF23, FGF23 ELISA kit (cat. no. CY-4000, KAINOS Laboratories, Inc., Tokyo, Japan, RRID: AB_2782966); serum 25(OH)D direct day ELISA Kit (cat. no. KR2108, Immundiagnostik AG, Bensheim, Germany, RRID: AB_2927705); PINP, rat/mouse PINP EIA (cat. no. AC-33F1, Immunodiagnostic Systems Ltd, Boldon, UK, RRID: AB_2801263); and CTX, Rat Laps EIA kit (cat. no. AC-06F1, Immunodiagnostic Systems Ltd., RRID: AB_2801265).

Hasegawa T., Tokunaga S., Yamamoto T., Sakai M., Hongo H., Kawata T, & Amizuka N. (2023). Evocalcet Rescues Secondary Hyperparathyroidism-driven Cortical Porosity in CKD Male Rats. Endocrinology, 164(4), bqad022.

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Mineralization Assay of PPU7 and HUCPVC Cells

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PPU7 was grown on a 12-well plate at an initial density of 3.16 × 10⁴ cells/cm². After incubation for 24 h, the mineralization medium was changed to the above mineralization medium containing 20% and 50% CM-PPU7 or CM-HUCPVC. The cells were cultured for up to 7 days. The mineralization was visualized by Alizarin Red S staining using the same method described above (see Section 2.5. Detection of mineralized nodules in HUCPVCs).
For the measurement of Ca concentration, each well on the plates was rinsed with PBS, and the calcium was dissolved in 0.5 mL of 0.5 N HCl with gentle rocking for 1 h. The calcium concentration in the eluate was determined using a calcium assay kit (Calcium E-test Wako, Wako Pure Chemical Industries, Ltd.) and by spectrophotometrically following the color development at 595 nm. All values were normalized against the cultivation area.

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Serum Biomarker Measurement Protocol

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The serum PTH level was measured using a Rat Intact PTH ELISA kit (Immutopics, Inc., San Clemente, CA). The serum calcium, phosphate and BUN levels were measured using an auto analyzer (Hitachi High-Technologies Corporation., Tokyo, Japan) or the following test kits: the Calcium E-test WAKO, the Phospha C-test WAKO, or the Blood Urea nitrogen B-test WAKO (Wako Pure Chemical Industries, Ltd., Osaka, Japan).

Kawata T., Tokunaga S., Murai M., Masuda N., Haruyama W., Shoukei Y., Hisada Y., Yanagida T., Miyazaki H., Wada M., Akizawa T, & Fukagawa M. (2018). A novel calcimimetic agent, evocalcet (MT-4580/KHK7580), suppresses the parathyroid cell function with little effect on the gastrointestinal tract or CYP isozymes in vivo and in vitro. PLoS ONE, 13(4), e0195316.

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Preparing Platelet-Rich Fibrin from Whole Blood

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WB samples were quickly centrifuged at 1500 rpm for 3 min to prepare plasma fraction, which were subjected to determine total free calcium levels using a commercial kit based on MXB method (Calcium E-test WAKO; Wako Pure Chemicals, Osaka, Japan).
Stored WB samples were then mixed intermittently with 200 μL (20 μL × 10 times) of 10% CaCl₂ solution and centrifuged by a Medifuge centrifugation system to prepare PRF. When lower amounts of CaCl₂ were added, PRF clots were less reproducibly prepared. When higher amounts of CaCl₂ were added intermittently, or when the optimal amount of CaCl₂ were added at once, PRF clots were never prepared (Kawase, unpublished observations).
The supernatant serum fractions were subjected to determine calcium and glucose levels as described above and using a commercial kit based on GOD method (Glucose CII-test WAKO; Wako Pure Chemicals). The serum fractions were also used to determine pH levels by pH indicators (MColorHast; EMD Millipore Corp., Billerica, MA, USA).

Isobe K., Suzuki M., Watanabe T., Kitamura Y., Suzuki T., Kawabata H., Nakamura M., Okudera T., Okudera H., Uematsu K., Nakata K., Tanaka T, & Kawase T. (2017). Platelet-rich fibrin prepared from stored whole-blood samples. International Journal of Implant Dentistry, 3, 6.

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