QuantiChrom™ Calcium Assay Kit
Protocol SDS


  • 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 96-well plate assay.
  • Simple and high-throughput. The procedure involves 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 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 lipid, protein and minerals such as magnesium, iron and zinc.


  • OD612nm


  • Biological, food and environment


  • All


  • 500 tests

Detection Limit

  • 0.08 mg/dL (20 μM)

Shelf Life

  • 12 months

More Details

  • CALCIUM is measured to monitor diseases of the 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 phenolsulphonephthalein 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 QuantiChromTM 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). ODSAMPLE

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

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

The whole blood sample concentration is computed as follows:

[Ca2+] = (ODSAMPLE - ODBLANK)/(ODSTANDARD - ODSAMPLE) x 10 x n (mg/dL)

where ODSAMPLE, ODBLANK, and ODSTANDARD 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.

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 QuantiChromTM 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.

For more detailed product information and questions, please feel free to Contact Us. Or for more general information regarding our assays, please refer to our General Questions.
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.

Li, Y., et al. (2019). Biofunctionalization of decellularized porcine aortic valve with OPG-loaded PCL nanoparticles for anti-calcification. RSC advances, 9(21), 11882-11893. Assay: Calcium in rats bone marrow.

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.

Tsolaki, E., Didierlaurent, L., Muller, E., Rottmar, M., Latif, N., Chester, A. H. & Bertazzo, S. (2019). Electron microscopy characterization of minerals formed in vitro by human bone cells and vascular smooth muscle cells. BioRxiv, 661785. Assay: Calcium in human bone 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.

Bouyoucos, I. A., Talwar, B. S., Brooks, E. J., Brownscombe, J. W., Cooke, S. J., Suski, C. D., & Mandelman, J. W. (2018). Exercise intensity while hooked is associated with physiological status of longline-captured sharks. Conservation physiology, 6(1), coy074. Assay: Calcium in shark plasma calcium.

Gurel Pekozer, G., Ramazanoglu, M., Schlegel, K. A., Kok, F. N., & Torun Kose, G. (2018). Role of STRO-1 sorting of porcine dental germ stem cells in dental stem cell-mediated bone tissue engineering. Artificial cells, nanomedicine, and biotechnology, 46(3), 607-618. Assay: Calcium in porcine teeth.

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.

Onal, M., Carlson, A. H., Thostenson, J. D., Benkusky, N. A., Meyer, M. B., Lee, S. M., & Pike, J. W. (2018). A Novel Distal Enhancer Mediates Inflammation-, PTH-, and Early Onset Murine Kidney Disease-Induced Expression of the mouse Fgf23 Gene. JBMR plus, 2(1), 31-46. Assay: Calcium in mice maxillary blood.

Roh, J. Y., & Kim, K. R. (2018). Anti-inflammatory effect of new calcium hydroxide paste containing silicon-substituted hydroxyapatite in lipopolysaccharide-stimulated macrophages. J Korean Soc Dent Hyg 18(4), 423-432. Assay: Calcium in plant.

Townsend, J. M., Zabel, T. A., Feng, Y., Wang, J., Andrews, B. T., Nudo, R. J. & Detamore, M. S. (2018). Effects of tissue processing on bioactivity of cartilage matrix-based hydrogels encapsulating osteoconductive particles. Biomedical Materials, 13(3), 034108. Assay: Calcium in rats bone marrow.

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.

Go, Y. Y., Kim, S. E., Cho, G. J., Chae, S. W., & Song, J. J. (2017). Differential effects of amnion and chorion membrane extracts on osteoblast-like cells due to the different growth factor composition of the extracts. PloS one, 12(8), e0182716. Assay: Calcium in human amniotic membrane.

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, E. C., Park, J., Kwon, I. K., Lee, S. W., Park, S. J., & Ahn, S. J. (2017). Static magnetic fields promote osteoblastic/cementoblastic differentiation in osteoblasts, cementoblasts, and periodontal ligament cells. Journal of periodontal & implant science, 47(5), 273-291. Assay: Calcium in human fetal osteoblast.

Paul, S., Gangwar, A., Bhargava, K., & Ahmad, Y. (2017). Deciphering Molecular Cascades In A Novel Acclimatization Strategy For Rapid Ascent To High Altitude. bioRxiv, 145342. Assay: Calcium in Sprague Dewley rats lung tissue/plasma.

Vanacker, N., Ollier, S., Beaudoin, F., Blouin, R., & Lacasse, P. (2017). Effect of inhibiting the lactogenic signal at calving on milk production and metabolic and immune perturbations in dairy cows. Journal of dairy science, 100(7), 5782-5791. Assay: Calcium in Holstein Cows serum.

Wang, F., Johnson, R. L., DeSmet, M. L., Snyder, P. W., Fairfax, K. C., & Fleet, J. C. (2017). Vitamin D Receptor-Dependent Signaling Protects Mice From Dextran Sulfate Sodium-Induced Colitis. Endocrinology, 158(6), 1951-1963. Assay: Calcium in mice intestinal cells.

Birgani, Z. T., van Blitterswijk, C. A., & Habibovic, P. (2016). Monolithic calcium phosphate/poly (lactic acid) composite versus calcium phosphate-coated poly (lactic acid) for support of osteogenic differentiation of human mesenchymal stromal cells. Journal of Materials Science: Materials in Medicine, 27(3), 54. Assay: Calcium in monolithic PLA/CaP composite.

Cai, T., Sun, D., Duan, Y., Wen, P., Dai, C., Yang, J., & He, W. (2016). WNT/beta-catenin signaling promotes VSMCs to osteogenic transdifferentiation and calcification through directly modulating Runx2 gene expression. Experimental cell research, 345(2), 206-217. Assay: Calcium in rats smooth muscles.

Corcoran, A., Bermudez, M. A., Seoane, S., Perez-Fernandez, R., Krupa, M., Pietraszek, A. & Marcinkowska, E. (2016). Biological evaluation of new vitamin D2 analogues. The Journal of steroid biochemistry and molecular biology, 164, 66-71. Assay: Calcium in mice serum.

Go, Y. Y., Kim, S. E., Cho, G. J., Chae, S. W., & Song, J. J. (2016). Promotion of osteogenic differentiation by amnion/chorion membrane extracts. Journal of applied biomaterials & functional materials, 14(2), 171-180. Assay: Calcium in human amniotic membrane.

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.

Kose, S et al (2016). Evaluation of biocompatibility of random or aligned electrospun polyhydroxybutyrate scaffolds combined with human mesenchymal stem cells. Turkish Journal of Biology, 40(2), 410-419. Assay: Calcium in human myscenchymal cells.

Mao, J., Shi, X., Wu, Y. B., & Gong, S. Q. (2016). Identification of specific hydroxyapatite {001} binding heptapeptide by phage display and its nucleation effect. Materials, 9(8), 700. Assay: Calcium in human teeth.

Onal, M., St. John, H. C., Danielson, A. L., & Pike, J. W. (2016). Deletion of the distal Tnfsf11 RL-D2 enhancer that contributes to PTH-mediated RANKL expression in osteoblast lineage cells results in a high bone mass phenotype in mice. Journal of Bone and Mineral Research, 31(2), 416-429. Assay: Calcium in mice osteoblast cells.

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.

Villa-Bellosta, R., Hamczyk, M. R., & Andres, V. (2016). Alternatively activated macrophages exhibit an anticalcifying activity dependent on extracellular ATP/pyrophosphate metabolism. American Journal of Physiology-Cell Physiology, 310(10), C788-C799. Assay: Calcium in mice smooth muscles.

Ballester-Lozano GF et al. (2015). Comprehensive biometric, biochemical and histopathological assessment of nutrient deficiencies in gilthead sea bream fed semi-purified diets. Br J Nutr. 114(5):713-26. Assay: calcium in fish 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.

Ou KL, et al (2010). Effects of the nanostructure and nanoporosity on bioactive nanohydroxyapatite/reconstituted collagen by electrodeposition. J Biomed Mater Res A. 92(3):906-12. Assay: Calcium in human stem 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.

Brand A, et al (2009). Calcium homeostasis is required for contact-dependent helical and sinusoidal tip growth in Candida albicans hyphae. Mol Microbiol. 71(5):1155-64. Assay: Calcium in cattle FBS.

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.

To find more recent publications, please click here.
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Please email or call 1-510-782-9988 x 2 to request assay service.
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QuantiChrom™ Calcium Assay Kit
Catalog No: DICA-500
Price: $319    Qty:
For orders of 10 or more kits, please call +1-510-782-9988x1 or email us for best pricing and/or bulk order.

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Shipment: Fedex Service
Delivery: 1-2 days (US), 3-6 days (Intl) Storage: 4°C

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