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Enzychrom creatine kinase assay kit

Manufactured by BioAssay Systems
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The EnzyChrom Creatine Kinase Assay Kit is a quantitative colorimetric assay for the measurement of creatine kinase activity in biological samples. The kit utilizes a proprietary enzyme-coupled reaction that results in a color development proportional to the creatine kinase concentration in the sample.

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Lab products found in correlation

Evos fl cell imaging system, by Thermo Fisher Scientific (2 mentions) Normal goat serum (ngs), by Vector Laboratories (2 mentions) Click it edu alexa fluor 594 imaging kit, by Thermo Fisher Scientific (2 mentions) Triton x 100, by Thermo Fisher Scientific (2 mentions) Paraformaldehyde (pfa), by Electron Microscopy Sciences (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Ldh kit, by Roche (1 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Colorimetric assay kit, by Abcam (1 mentions) Bca assay, by Thermo Fisher Scientific (1 mentions) Synergy plate reader, by Agilent Technologies (1 mentions) Transaminase cii test wako, by Fujifilm (1 mentions) Ripa buffer, by Nacalai Tesque (1 mentions) Mouse il 6 elisa kit, by RayBiotech (1 mentions) Antibiotics and antimycotic, by Thermo Fisher Scientific (1 mentions) Ctrl sirna, by Santa Cruz Biotechnology (1 mentions) Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)

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EnzyChrom Kinase Assay Kit (3 citations) EnzyChrom Creatine Kinase Assay (2 citations)

13 protocols using enzychrom creatine kinase assay kit

1

Quantification of Cell Death Markers

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The severity of heart tissue damage or cell death with cell membrane disruption was determined by the release of intracellular LDH (a marker for cell death) and/or CK (a specific marker for myocardial tissue damage) to the preservation solution or serum. LDH release was quantitated by the LDH assay using a cytotoxicity detection (LDH) kit (Roche Applied Science, Laval, QC, Canada) following manufacturers’ protocols. In cultured cells, LDH release in the preservation solution was presented as a percentage of positive control (cells incubating with 2% Triton X-100). In sera or isolated hearts, LDH was expressed as the absorbance at 490 nm (OD490). Serum CK level was measured as a unit per liter (U/L) using Enzychrom Creatine Kinase Assay kit (BioAssay System, Hayward, CA, USA).
Gao S., Guan Q., Chafeeva I., Brooks D.E., Nguan C.Y., Kizhakkedathu J.N, & Du C. (2015). Hyperbranched Polyglycerol as a Colloid in Cold Organ Preservation Solutions. PLoS ONE, 10(2), e0116595.
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2

Creatine Kinase Activity in Mouse Brain Mitochondria

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The EnzyChrom Creatine Kinase Assay Kit (BioAssay Systems) was applied to measure activity in mouse brain mitochondria (as above). Two brains each from 12 month-old mice were pooled to provide enough mitochondria and normalized for total protein concentration. Mitochondria were incubated with 0 or 0.5 mM H2O2 at room temperature for 20 min prior to assay start. The creatine kinase-dependent catalytic conversion of creatine phosphate and ADP to creatine and ATP was quantified indirectly by measuring NADPH at 340 nm. ATP produced by the reaction phosphorylates glucose to glucose-6-phosphate (G6P) by hexokinase, which is oxidized by NADP+ in the presence of G6P-dehydrogenase, yielding NADPH. Rates in uM/min were calculated for 3 independent experiments done in triplicate.
El Kodsi D.N., Tokarew J.M., Sengupta R., Lengacher N.A., Chatterji A., Nguyen A.P., Boston H., Jiang Q., Palmberg C., Pileggi C., Holterman C.E., Shutinoski B., Li J., Fehr T.K., LaVoie M.J., Ratan R.R., Shaw G.S., Takanashi M., Hattori N., Kennedy C.R., Harper M.E., Holmgren A., Tomlinson J.J, & Schlossmacher M.G. (2023). Parkin coregulates glutathione metabolism in adult mammalian brain. Acta Neuropathologica Communications, 11, 19.
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3

Assaying Cell Proliferation and Myotube Differentiation

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To compare rates of cell proliferation, incorporation of the nucleoside analog 5-ethynyl-2’-deoxyuridine (EdU) was measured using the Click-iT EdU Alexa Fluor 594 Imaging Kit per manufacturer’s instructions (Life Technologies). To compare myotube differentiation, cells were stained with anti-sarcomeric myosin (MF20-s, DSHB, Iowa City, IA, USA, 1:1) after fixing with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA), permeabilizing with 0.1% Triton X-100 (Fisher Scientific, Hampton, NH, USA), and blocking with 5% normal goat serum (Vector Laboratories, Burlingame, CA, USA). For both proliferation and differentiation, images were acquired using an EVOS FL Cell Imaging System equipped with a 20×/0.45 NA objective (Life Technologies). Images were analyzed using ImageJ (National Institutes of Health, Bethesda, MD, USA). The myofusion index was calculated as the percentage of nuclei that were found in MF20-positive fibers. As an additional measure of differentiation, creatine kinase (CK) activity was compared between control and Mss51-disrupted cells using the EnzyChrom Creatine Kinase Assay Kit (BioAssay Systems, Hayward, CA, USA) following the manufacturer’s instructions.
Moyer A.L, & Wagner K.R. (2015). Mammalian Mss51 is a Skeletal Muscle-Specific Gene Modulating Cellular Metabolism. Journal of Neuromuscular Diseases, 2(4), 371-385.
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4

Serum Creatine Kinase Assay

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Serum creatine kinase (CK) activity was determined using an EnzyChrom™ Creatine Kinase Assay Kit (BioAssay Systems) according to the manufacturer's instructions. Briefly, 10 µl of mouse serum was added to 100 µl of reconstituted reagent into a 96‐well plate. To calibrate the data, calibrator, 10 µl, in 100 µl of water was used. Samples were incubated at room temperature. The rate of change in NADPH absorbance at 340 nm, which is proportionate to the CK activity, was measured after 20 and 40 min using Synergy H1 Hybrid Multi‐Mode Microplate Reader (Vermont, USA). CK activity was calculated by the following equation: [(A 340)4min–(A 340)20mm]/[(A 340)calibrator–[(A 340)blank] × 150.
Ramani Sattiraju S., Jama A., Alshudukhi A.A., Edward Townsend N., Reynold Miranda D., Reese R.R., Voss A.A, & Ren H. (2020). Loss of membrane integrity drives myofiber death in lipin1‐deficient skeletal muscle. Physiological Reports, 8(20), e14620.
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5

Quantifying Cell Proliferation and Differentiation

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To compare rates of cell proliferation, incorporation of the nucleoside analog 5-ethynyl– 2′-deoxyuridine (EdU) was measured using the Click-iT EdU Alexa Fluor 594 Imaging Kit per manufacturer’s instructions (Life Technologies). To compare myotube differentiation, cells were stained with anti-sarcomeric myosin (MF20-s, DSHB, Iowa City, IA, USA, 1:1) after fixing with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA), permeabilizing with 0.1% Triton X-100 (Fisher Scientific, Hampton, NH, USA), and blocking with 5% normal goat serum (Vector Laboratories, Burlingame, CA, USA). For both proliferation and differentiation, images were acquired using an EVOS FL Cell Imaging System equipped with a 20×/0.45 NA objective (Life Technologies). Images were analyzed using ImageJ (National Institutes of Health, Bethesda, MD, USA). The myofusion index was calculated as the percentage of nuclei that were found in MF20-positive fibers. As an additional measure of differentiation, creatine kinase (CK) activity was compared between control and Mss51-disrupted cells using the EnzyChrom Creatine Kinase Assay Kit (BioAssay Systems, Hayward, CA, USA) following the manufacturer’s instructions.
Moyer A.L, & Wagner K.R. (2015). Mammalian Mss51 is a Skeletal Muscle-Specific Gene Modulating Cellular Metabolism. Journal of Neuromuscular Diseases, 2(4), 371-385.
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6

Myoblast Differentiation and Silencing

Cited 1 time
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Mouse C2C12 myoblasts were cultured in growth medium (Dulbecco's Modified Eagle's Medium (DMEM, Life Technologies) containing 20% fetal bovine serum (FBS, Gibco) and antibiotics. Human KM155 myoblasts were cultured in growth medium (equal volume mixture of Hamm’s F10 media with 20% FBS and Promocell Skeletal Muscle Cell Growth Medium). The C2C12 and KM155 myoblasts were maintained at 37°C in a 5% CO2 humidified atmosphere and were differentiated by replacing the growth medium with differentiation medium (DMEM with 2% horse serum) [6 (link)]. For silencing experiments, control small interfering RNA (Ctrl siRNA) or SPARC siRNA was transfected twice, 36 h and 12 h before inducing differentiation. MicroRNA mimics and inhibitors for miR-451a and miR-144-3p (Sigma Aldrich) were transfected twice, 36 h and 12 h before induction of differentiation at a final concentration of 50 nM using Lipofectamine 2000 (Life Technologies). Creatine kinase (CK) activity was determined in cell lysates using the EnzyChrom creatine kinase assay kit (BioAssay Systems) following the manufacturer's protocol.
Munk R., Martindale J.L., Yang X., Yang J.H., Grammatikakis I., Di Germanio C., Mitchell S.J., de Cabo R., Lehrmann E., Zhang Y., Becker K.G., Raz V., Gorospe M., Abdelmohsen K, & Panda A.C. (2019). Loss of miR-451a enhances SPARC production during myogenesis. PLoS ONE, 14(3), e0214301.
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7

Biomarker Assays for Organ Injury

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Circulating biomarkers of organ injury were assayed with the following commercially available kits: uric acid with a Uric Acid Colorimetric/Fluorometric Assay kit; aspartate aminotransferase activity with a Colorimetric Assay kit from Biovision (Milpitas, California); Creatine kinase with an EnzyChrom Creatine Kinase Assay kit from BioAssay System (Hayward, California); and troponin I with a Mouse High Sensitivity Cardiac Troponin I Elisa kit from MyBioSource (San Diego, California).
He Q., Wang F., Ryan T.D., Chalasani M, & Redington A.N. (2020). Repeated Remote Ischemic Conditioning Reduces Doxorubicin-Induced Cardiotoxicity. JACC: CardioOncology, 2(1), 41-52.
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8

Measuring Myogenic Differentiation

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Primary myoblasts from wild-type and lipin1Myf5cKO mice were plated in 6-well plates. Upon confluency, cell culture medium was replaced using differentiation medium containing high glucose DMEM supplemented with 5% horse serum to induce myogenic differentiation. Media was changed every two days. At day 6, the media from the differentiated primary cells were collected and prepared for creatine kinase activity measurement using EnzyChrom™ Creatine Kinase Assay kit (BioAssay Systems). The assay was performed in triplicate and results normalized to total protein levels using a BCA assay (Thermo Fisher).
Jama A., Alshudukhi A.A., Burke S., Dong L., Kamau J.K., Voss A.A, & Ren H. (2023). Lipin1 plays complementary roles in myofiber stability and regeneration in dystrophic muscles. The Journal of physiology, 601(5), 961-978.
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9

Quantifying Cellular Creatine Kinase

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Cellular creatine kinase levels were analyzed using the EnzyChrom™ Creatine Kinase Assay Kit (BioAssay Systems, Hayward, CA, USA), according to manufacturers' instructions.
Ahmad S.S., Lim J.H., Ahmad K., Chun H.J., Hur S.J., Lee E.J, & Choi I. (2024). Targeting myostatin using quercetin as a media supplement to improve myogenesis for cultured meat production: An in silico and in vitro study. Current Research in Food Science, 8, 100678.
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10

Quantitative Creatine Kinase Assay

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CK levels in serum were determined using Enzychrom Creatine Kinase Assay kit (Bioassay Systems, Hayward, CA) following manufacturer's instructions. The kit uses an enzyme-coupled reaction in which creatine phosphate and adenosine diphosphate (ADP) are converted to creatine and adenosine triphosphate (ATP) by CK. The ATP generated in the process is used by hexokinase in phosphorylating glucose to glucose-6-phosphate. The latter is subsequently oxidized by nicotinamide adenine dinucleotide phosphate (NADP), in the presence of glucose-6-phosphate dehydrogenase and produces reduced NADP (NADPH). The produced NADPH is proportional to the CK activity in the samples and was quantified by measuring the absorbance at 340 nm using Biotek Synergy plate reader (Biotek, Winooski, VT).
Dutta D., Xu J., Dirain M.L, & Leeuwenburgh C. (2014). Calorie restriction combined with resveratrol induces autophagy and protects 26-month-old rat hearts from doxorubicin-induced toxicity. Free radical biology & medicine, 74, 252-262.
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