QuantiChrom™ Calcium Assay Kit

Protocol SDS

Application

• For quantitative determination of calcium ion Ca²⁺ and evaluation of drug effects on calcium metabolism.

Key Features

• Sensitive and accurate. Use as little as 5 µL samples. Linear detection range 0.08 mg/dL (20 µM) to 20 mg/dL (5 mM) Ca²⁺ in a 96-well plate assay.
• Simple and high-throughput. The procedure involves the addition of a single working reagent and incubation for 3 min. Can be readily automated as a high-throughput assay for thousands of samples per day.
• Improved reagent stability and versatility. The optimized formulation has greatly enhanced the reagent and signal stability. Cuvet or 96-well plate assay.
• Low interference in biological samples. No pretreatments are needed. Assays can be directly performed on raw biological samples i.e., in the presence of lipids, protein,s, and minerals such as magnesium, iron, and zinc.

Method

• OD612nm

Samples

• Biological, food, and environment

Species

• All

Procedure

• 3 min

Size

• 500 tests

Detection Limit

• 0.08 mg/dL (20 µM)

Shelf Life

• 12 months

More Details

CALCIUM is measured to monitor diseases of bone or calcium regulation disorders. Increased calcium levels in serum are reported in hyperparathyroidism, metastatic bone lesions, and hypervitaminosis, while decreased levels are observed in hypoparathyroidism, nephrosis, rickets, steatorrhea, nephritis, and calcium-losing syndromes. Urinary calcium levels aid the clinician in understanding how the kidneys handle calcium in certain diseases of the parathyroid gland. Urinary calcium levels are also essential in the medical evaluation of kidney stones. Simple, direct, and automation-ready procedures for measuring calcium concentration in biological samples are becoming popular in Research and Drug Discovery. BioAssay Systems calcium assay kit is designed to measure calcium directly in biological samples without any pretreatment. A phenol sulphone phthalein dye in the kit forms a very stable blue-colored complex specifically with free calcium. The intensity of the color, measured at 612 nm, is directly proportional to the calcium concentration in the sample. The optimized formulation minimizes any interference by substances such as magnesium, lipid, protein, and bilirubin.

We have whole blood samples. Does your assay work?

Yes, our QuantiChrom^TM Calcium Assay Kit (DICA-500) can be used on whole blood samples. To correct for interference in the sample matrix, two internal standard methods have been validated. Protocol A is quicker whereas Protocol B is slightly more involved, but requires less sample and is, thus, recommended for customer’s that have a limited quantity of sample. Additionally, protocol B requires less Reagent because each sample requires one well rather than three separate wells per sample. Please note that 20 mM EDTA is needed for this experiment and is not provided. The customer should prepare this solution, or is available for purchase upon request.

PROTOCOL A: 3 Separate wells needed for each sample

A1. Whole Blood samples require an internal standard and need three separate reactions: 1) Sample plus Standard 2) Sample alone and 3) Sample Blank. For the internal standard prepare 250 µL 10 mg/dL Ca²⁺ Standard by mixing 125 µL 20 mg/dL Standard and 125 µL dH₂O.

Transfer 5 µL whole blood sample to three separate wells. Add 5 µL of 10 mg/dL Ca²⁺ to the 1) Sample plus Standard well, 5 µL dH₂O to 2) Sample alone well and 5 µL 20 mM EDTA to 3) sample Blank well.

A2. Add 200 µL Working Reagent and tap lightly to mix. Note: If any particulates or turbidity are seen pipette up and down to dissolve.

A3. Incubate 3 min at room temperature and read optical density at 570-650 nm (peak absorbance at 612 nm).

PROTOCOL B: 1 Well needed for each sample

B1. Dilute standard to 10 mg/dL Ca²⁺ by mixing 125 µL 20 mg/dL Standard and 125 µL dH₂O.

B2. Transfer 5 µL whole blood sample to a well.

B3. Add 200 µL Working Reagent and tap lightly to mix. Note: If any particulates are seen pipette up and down to dissolve.

B4. Incubate 3 min at room temperature and read optical density at 570-650 nm (peak absorbance at 612 nm). OD_SAMPLE

B5. Carefully transfer 5 µL of 10 mg/dL standard to the sample well from step 2. Tap plate to mix. Repeat Step 4. OD_STANDARD

B6. Add 5 µL of 20 mM EDTA to the same well from step 2. Tap plate to mix. Repeat step 4. OD_BLANK

CALCULATION
The whole blood sample concentration is computed as follows:

[Ca²⁺] = (OD_SAMPLE – OD_BLANK)/(OD_STANDARD – OD_SAMPLE) x 10 x n (mg/dL)

where OD_SAMPLE, OD_BLANK, and OD_STANDARD are the OD readings of the Sample, Sample Blank, and the Sample plus Standard respectively, 10 is the concentration of the standard in mg/dL, and n is the sample dilution factor. If the calculated calcium concentration is greater than 10 mg/dL, dilute sample in dH₂O and repeat assay. Multiply result by the dilution factor n.

EXAMPLE
One human blood sample was assayed using the two methods. The Ca²⁺ concentration was 8.48 mg/dL using Protocol A and 8.38 mg/dL using Protocol B.

Is the calcium assay compatible with acids?

Yes, our QuantiChrom^TM Calcium Assay is compatible with acids, such as 0.5 M HCl.

Do you know if phosphate in the sample will interfere with the calcium assay or if calcium in the sample will interfere with the phosphate assay?

Phosphate (at least up to 30 mM) in the sample does not interfere with the calcium assay.

Does freezing of serum have any impact on serum Ca compared to using fresh serum?

Our assay kit measures the total calcium content of samples. In serum about half of the calcium is free (“ionized”) and the other half is bound to proteins, especially albumin (~40%) or anions (~10%). Repeated freeze-thaw cycles may cause precipitation of proteins which would alter the outcome of the assay. We recommend avoiding repeated freeze thaw cycles for serum samples.

Harb, S. V., et al. (2020). Hydroxyapatite and β-TCP modified PMMA-TiO2 and PMMA-ZrO2 coatings for bioactive corrosion protection of Ti6Al4V implants. Materials Science and Engineering: C, 116, 111149. Assay: Calcium in human fetal osteoblast cells.

Kohart, N. A., et al (2020). Parathyroid hormone-related protein promotes bone loss in T-cell leukemia as well as in solid tumors. Leukemia & Lymphoma, 61(2), 409-419. Assay: Calcium in mouse plasma and bone cell media.

Su, Z., et al. (2020). Celastrol attenuates arterial and valvular calcification via inhibiting BMP2/Smad1/5 signalling. Journal of Cellular and Molecular Medicine, 24(21), 12476-12490. Assay: Calcium in pig aorta cells.

Zhou, J., et al. (2020). HDAC1-mediated deacetylation of LSD1 regulates vascular calcification by promoting autophagy in chronic renal failure. Journal of Cellular and Molecular Medicine, 24(15), 8636-8649. Assay: Calcium in rat cells and tissue.

Choe, N., et al. (2020). The microRNA miR-134-5p induces calcium deposition by inhibiting histone deacetylase 5 in vascular smooth muscle cells. Journal of Cellular and Molecular Medicine, 24(18), 10542-10550. Assay: Calcium in rat cells.

Xu, T.-H., et al. (2020). OGT knockdown counteracts high phosphate-induced vascular calcification in chronic kidney disease through autophagy activation by downregulating YAP. Life Sciences, 261, 118121. Assay: Calcium in rat cells and tissue.

Lee, J., et al. (2020). Metformin, resveratrol, and exendin-4 inhibit high phosphate-induced vascular calcification via AMPK-RANKL signaling. Biochemical and Biophysical Research Communications, 530(2), 374-380. Assay: Calcium in rat cells.

Zhu, X., et al. (2020). Reversal of phosphate-induced ORAI1 expression, store-operated Ca2+ entry and osteogenic signaling by MgCl2 in human aortic smooth muscle cells. Biochemical and Biophysical Research Communications, 523(1), 18-24. Assay: Calcium in human cells.

Ern, C., et al. (2019). Effects of prostaglandin E2 and D2 on cell proliferation and osteogenic capacity of human mesenchymal stem cells. Prostaglandins, Leukotrienes and Essential Fatty Acids, 151, 1-7. Assay: Calcium in human mesenchymal cells.

Li, Y., et al. (2021). MiR155 modulates vascular calcification by regulating Akt-FOXO3a signalling and apoptosis in vascular smooth muscle cells. Journal of Cellular and Molecular Medicine, 25(1), 535-548. Assay: Calcium in mice serum and cells.

Villa-Bellosta, R. (2020). Dietary magnesium supplementation improves lifespan in a mouse model of progeria. EMBO Molecular Medicine, 12(10). Assay: Calcium in mouse tissue, plasma, and cells.

Lee, J., et al (2020). Bioactive membrane immobilized with lactoferrin for modulation of bone regeneration and inflammation. Tissue Engineering Part A, 26(23-24), 1243-1258. Assay: Calcium in human adipose stem cells.

Lai, C.H., et al (2020). Data supporting the effects of xanthine derivative KMUP-3 on vascular smooth muscle cell calcification and abdominal aortic aneurysm in mice. Data in Brief, 30, 105550. Assay: Calcium in rat cells.

Zhu, X., et al. (2021). Vasopressin-stimulated ORAI1 expression and store-operated Ca2+ entry in aortic smooth muscle cells. Journal of Molecular Medicine. Assay: Calcium in human muscle cells.

Wen, M., et al (2020). Local delivery of dual micrornas in trilayered electrospun grafts for vascular regeneration. ACS Applied Materials & Interfaces, 12(6), 6863-6875. Assay: Calcium in human cells.

Srinivasan, A., et al (2021). Comparative craniofacial bone regeneration capacities of mesenchymal stem cells derived from human neural crest stem cells and bone marrow. ACS Biomaterials Science & Engineering, 7(1), 207-221. Assay: Calcium in artificial cell scaffold.

Kim, H., et al. (2020). Chemerin treatment inhibits the growth and bone invasion of breast cancer cells. International Journal of Molecular Sciences, 21(8). Assay: Calcium in mouse serum.

Park, S., et al (2019). Suppression of spry4 promotes osteogenic differentiation and bone formation of mesenchymal stem cell. Tissue Engineering. Part A, 25(23-24), 1646-1657. Assay: Calcium in human adipose stem cells.

Toan, N. K., et al. (2021). Choline acetyltransferase induces the functional regeneration of the salivary gland in aging samp1/kl -/- mice. International Journal of Molecular Sciences, 22(1). Assay: Calcium in mouse tissue.

Rimondi, E., et al. (2021). Role of vitamin D in the pathogenesis of atheromatosis. Nutrition, Metabolism, and Cardiovascular Diseases: NMCD, 31(1), 344-353. Assay: Calcium in mouse serum.

Choe, N., et al. (2020). Mir-27a-3p targets atf3 to reduce calcium deposition in vascular smooth muscle cells. Molecular Therapy – Nucleic Acids, 22, 627-639. Assay: Calcium in rat cells.

Poetsch, F., et al. (2020). Role of sgk1 in the osteogenic transdifferentiation and calcification of vascular smooth muscle cells promoted by hyperglycemic conditions. International Journal of Molecular Sciences, 21(19). Assay: Calcium in human primary cells.

Komuro, H., et al (2020). Cardiomyocyte uptake mechanism of a hydroxyapatite nanoparticle mediated gene delivery system. Beilstein Journal of Nanotechnology, 11, 1685-1692. Assay: Calcium in artificial nanoparticles.

Lanigan, L. G., et al. (2021). In vivo tumorigenesis, osteolytic sarcomas, and tumorigenic cell lines from transgenic mice expressing the human t-lymphotropic virus type 1 (HTLV-1) tax viral oncogene. The American Journal of Pathology, 191(2), 335-352. Assay: Calcium in cell media.

Ma, C., et al. (2020). Circrna cdr1as promotes pulmonary artery smooth muscle cell calcification by upregulating camk2d and cnn3 via sponging mir-7-5p. Molecular Therapy. Nucleic Acids, 22, 530-541. Assay: Calcium in human cells.

Maleitzke, T., et al. (2020). Proinflammatory and bone protective role of calcitonin gene-related peptide alpha in collagen antibody-induced arthritis. Rheumatology (Oxford, England). Assay: Calcium in mouse serum.

Munoz, M. F.,et al. (2020). Effect of age and lipoperoxidation in rat and human adipose tissue-derived stem cells. Oxidative Medicine and Cellular Longevity, 2020, 6473279. Assay: Calcium in cell media.

Luong, T. T. D., et al (2021). Acid sphingomyelinase promotes SGK1-dependent vascular calcification. Clinical Science (London, England: 1979), 135(3), 515-534. Assay: Calcium in mouse cells or tissue.

Cruz, A.C.C. et al (2019). Retinoic acid increases the effect of bone morphogenetic protein type 2 on osteogenic differentiation of human adipose-derived stem cells. Journal of Applied Oral Science 27:e20180317. Assay: Calcium in human mesenchymal stem cells.

Schanstra, J. P.et al. (2019). Systems biology identifies cytosolic PLA2 as a target in vascular calcification treatment. JCI insight, 4(10) pii: 125638. Assay: Calcium in human plasma.

Schelski, N., Luong, T. T., Lang, F., Pieske, B., Voelkl, J., & Alesutan, I. (2019). SGK1-dependent stimulation of vascular smooth muscle cell osteo-/chondrogenic transdifferentiation by interleukin-18. Pflugers Archiv-European Journal of Physiology, 471(6):889-899. Assay: Calcium in human aortic smooth muscle cells.

Bora, S. A., Kennett, M. J., Smith, P. B., Patterson, A. D., & Cantorna, M. T. (2018). The gut microbiota regulates endocrine vitamin D metabolism through fibroblast growth factor 23. Frontiers in immunology, 9, 408. Assay: Calcium in mice cells.

Kagi, L., Bettoni, C., Pastor-Arroyo, E. M., Schnitzbauer, U., Hernando, N., & Wagner, C. A. (2018). Regulation of vitamin D metabolizing enzymes in murine renal and extrarenal tissues by dietary phosphate, FGF23, and 1, 25 (OH) 2D3. PloS one, 13(5), e0195427. Assay: Calcium in mice spot urine.

Ma, G. T., Lee, S. K., Park, K. K., Park, J., Son, S. H., Jung, M., & Chung, W. Y. (2018). Artemisinin-Daumone Hybrid Inhibits Cancer Cell-Mediated Osteolysis by Targeting Cancer Cells and Osteoclasts. Cellular Physiology and Biochemistry, 49(4), 1460-1475. Assay: Calcium in mice serum.

Vollersen, N., Hermans-Borgmeyer, I., Cornils, K., Fehse, B., Rolvien, T., Triviai, I. & Yorgan, T. A. (2018). High Bone Turnover in Mice Carrying a Pathogenic Notch2 Mutation Causing Hajdu-Cheney Syndrome. Journal of Bone and Mineral Research, 33(1), 70-83. Assay: Calcium in mice serum.

Aimaiti, A., Maimaitiyiming, A., Boyong, X., Aji, K., Li, C., & Cui, L. (2017). Low-dose strontium stimulates osteogenesis but high-dose doses cause apoptosis in human adipose-derived stem cells via regulation of the ERK1/2 signaling pathway. Stem cell research & therapy, 8(1), 282. Assay: Calcium in human bone marrow.

BARINDA, A. J., Ikeda, K., Hirata, K. I., & Emoto, N. (2017). Macrophages Highly Express Carbonic Anhydrase 2 and Play a Significant Role in Demineralization of the Ectopic Calcification. Kobe Journal of Medical Sciences, 63(2), E45. Assay: Calcium in culture plate fibronectin.

Hu, K., Sun, H., Gui, B., & Sui, C. (2017). Gremlin-1 suppression increases BMP-2-induced osteogenesis of human mesenchymal stem cells. Molecular medicine reports, 15(4), 2186-2194. Assay: Calcium in human bone marrow.

Kim, W., et al. (2016). Calcium-sensing receptor promotes breast cancer by stimulating intracrine actions of parathyroid hormone-related protein. Cancer research, 76(18), 5348-5360. Assay: Calcium in mice serum.

Villa-Bellosta, R., Gonzalez-Parra, E., & Egido, J. (2016). Alkalosis and dialytic clearance of phosphate increases phosphatase activity: a hidden consequence of hemodialysis. PloS one, 11(7), e0159858. Assay: Calcium in human plasma.

Chaumet-Riffaud P, et al (2010). Synthesis and application of lactosylated, 99mTc chelating albumin for measurement of liver function. Bioconjug Chem. 21(4):589-96. Assay: Calcium in mice liver tissue.

Jung GY, et al (2010). Effects of HA released calcium ion on osteoblast differentiation. J Mater Sci Mater Med. 21(5):1649-54. Assay: Calcium in mouse 3T3 cell.

Ponda MP,et al (2010). Moderate kidney disease inhibits atherosclerosis regression. Atherosclerosis.210(1):57-62. Assay: Calcium in mice serum.

Zarjou A, et al (2010). Ferritin ferroxidase activity: a potent inhibitor of osteogenesis. J Bone Miner Res. 25(1):164-72. Assay: Calcium in human osteoblasts cell.

Koreckij T, et al (2009). Dasatinib inhibits the growth of prostate cancer in bone and provides additional protection from osteolysis. Br J Cancer.101(2):263-8. Assay: Calcium in mice serum.

Villa-Bellosta R, Sorribas V (2009). Phosphonoformic acid prevents vascular smooth muscle cell calcification by inhibiting calcium-phosphate deposition. Arterioscler Thromb Vasc Biol. 29(5):761-6. Assay: Calcium in rat muscle cell.

Chanda, D et al (2008). Systemic osteoprotegerin gene therapy restores tumor-induced bone loss in a therapeutic model of breast cancer bone metastasis. Mol Ther. 16(5):871-8. Assay: Calcium in mouse serum.

He X, et al (2008). Effect of grafting RGD and BMP-2 protein-derived peptides to a hydrogel substrate on osteogenic differentiation of marrow stromal cells. Langmuir. 24(21):12508-16. Assay: Calcium in human marrow cell.

Henderson JA, et al (2008). Concurrent differentiation of marrow stromal cells to osteogenic and vasculogenic lineages. Macromol Biosci. 8(6):499-507. Assay: Calcium in human marrow cell.

Warotayanont R, et al (2008). Leucine-rich amelogenin peptide induces osteogenesis in mouse embryonic stem cells. Biochem Biophys Res Commun. 367(1):1-6. Assay: Calcium in mouse stem cell.

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If you or your labs do not have the equipment or scientists necessary to run this assay, BioAssay Systems can perform the service for you.

– Fast turnaround
– Quality data
– Low cost

Please email or call 1-510-782-9988 x 2 to discuss your projects.