The scaffolds with cells at different time points were lysed using M-PER Mammalian Protein Extraction Reagent (78501, Thermo Scientific) supplemented with protease inhibitor and phosphatase inhibitor cocktail (1:100) (Thermofisher, 78446). The lysed cell solution was centrifuged (4 °C, 14,000×g, 10 min), and the extracted protein supernatant was stored at −20 °C for further measurement of total protein and CK quantification. CK activity was calculated corresponding to the manufacturer’s procedure (MAK116, Sigma Aldrich). Briefly, 10 µL of protein extract was added to a 96-well plate, and 100 mL of reconstituted reagent and incubated at 37 °C. The incubated samples on 96-well plate was measured at 340 nm at 20 and 40 min using a microplate reader (TECAN, infinite M200pro). The final CK activity was normalised by total protein content, which was determined by BCA protein assay (23225, Thermo Scientific). CK activity was calculated using the absorbance of 20 and 40 min per the protocol-instructed formula, as shown in Eq. (1 ) below27 (link). Data were analysed using GraphPad Prism (software version 9.5.0). Statistical significance was determined through one-way ANOVA with Tukey’s multiple comparisons tests.
Mak116
Manufactured by Merck Group
Sourced in United States
The MAK116 is a laboratory equipment product. It is designed to perform specific functions in a laboratory setting. No further details about its core function or intended use can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Mak066, by Merck Group (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Infinite m200 pro, by Tecan (1 mentions)
S311 10, by Dojindo Laboratories (1 mentions)
Mak085, by Merck Group (1 mentions)
Quantikine human il 6, by R&D Systems (1 mentions)
A113 2, by Nanjing Jiancheng (1 mentions)
Mak052, by Merck Group (1 mentions)
Mak055, by Merck Group (1 mentions)
A111 2, by Nanjing Jiancheng (1 mentions)
Mak077, by Merck Group (1 mentions)
Mak080, by Merck Group (1 mentions)
Mak006, by Merck Group (1 mentions)
Lb 940, by Berthold Technologies (1 mentions)
13 protocols using mak116
1
Creatine Kinase Activity Measurement
2
Serum Creatine Kinase Quantification
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Quantification of serum creatine kinase activity was performed with a commercial kit (MAK116; Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions.
3
Oxidative Stress Biomarkers in Ischemic Injury
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Blood samples were collected at 24 h after reperfusion and plasma was isolated. The creatine kinase (CK, MAK116, Sigma) and lactate dehydrogenase (LDH, MAK066, Sigma) level in plasma were measured according to the manufacturer's instructions. Ventricles were crushed to a powder using liquid nitrogen and homogenized in saline with the weight/volume ratio of 1:10. After centrifuging for 10 min at 3500 rpm, the supernatants were withdrawn for SOD (S311–10, Dojindo Molecular Technologies) and MDA (MAK085, Sigma) measurement according to the manufacturer's instructions. Bradford protein assay was performed to determine the protein concentration.
4
Quantifying Cardiac Biomarkers in Mice
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Mouse serum samples were collected and stored at −80°C. The concentration of CK-MB (MAK116, Sigma, St. Louis) and cTnT (MAB18742-100, Novus Biologicals) in the serum of mice was measured via enzyme-linked immunosorbent assay kits in accordance with the manufacturer’s instructions. Briefly, samples were seeded in 96-well plates provided by the ELISA kits. Following testing was performed as per the protocols contained in the ELISA kit.
5
Quantification of Plasma CK and IL-6
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Quantification of plasma CK activity was performed with a commercial kit (MAK116, Sigma-Aldrich, St. Louis, USA) and quantification of plasma IL-6 concentration was performed with a commercial ELISA kit (Human IL-6 Quantikine, R&D Systems, Minneapolis, USA) in accordance with the manufacturer’s instructions.
6
Creatine Kinase Activity Assay
7
Creatine Kinase Activity Assay
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Creatine kinase activity was measured following the manufacturer’s instructions of a commercially available kit (Sigma Aldrich MAK116). Blood was collected from the tail, incubated at RT for 30 min then spun at 1,500 × g at 4 °C for 30 min. The clear portion was removed (serum) and the clot was discarded. Samples were aliquoted and stored at −80 °C until needed at which point they were thawed on ice and diluted 1:10 in dH2O prior to assay execution.
8
Plasma Creatine Kinase Estimation
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Blood samples were collected from the tail vein of all animals to estimate the plasma level of creatine kinase (CK) as a biomarker for myopathy and muscle breakdown (Jansone et al. 2016 ) using a commercial kit (MAK116) from Sigma-Aldrich (St. Louis, MO, USA).
9
Myotoxic Activity of Venom in Mice
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Venom test doses (4 μg crude venom) were prepared in a constant volume of 50 μL and injected into the right gastrocnemius muscle of five mice (20±2g) in test group. Control mice were injected 50 μL saline solution. In the present study, myotoxic activity of venom was found only after 6 h of injection of venom. According to WHO guidelines, creatine kinase (CK) was measured after 3 h of venom administration but CK activity of plasma was not observed at that time in this study. Therefore, blood sample was collected after 6 h injection according to Hardy et al. (2014) method. After 6 h, the blood sample was drawn using cardiac puncture and centrifuged at 3000 rpm for 10 min. The serum obtained by centrifugation was diluted with saline at a 1:10 ratio. Then, CK activity of serum was measured in 10 μL of serum according to the creatine kinase activity assay kit protocol (MAK 116, Sigma-Aldrich-Lot 116BJ10A23) (Hardy et al., 2014 ; World Health Organization, 2017 ).
10
Serum CK Activity Measurement in Mice
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Blood samples (~100 μL/mouse) were collected from the tail vein of mice at rest. Then the same group of mice were subjected to a 30 min running protocol at 18 m/min, 15° downhill. Right after completion of the running protocol, the blood samples were collected again. The serum was collected from supernatant after centrifuging the blood samples at 2000 g for 10 min at 4 °C. Freshly made serum samples were used to measure CK activity following the protocol provided by Sigma (MAK–116). Briefly, for each reaction, 10 μL of serum was mixed with 100 μL assay buffer; the mixture was then loaded onto a 96-well plate for recording the absorbance at 340 nm at time intervals of 5 min up to 40 min in a plate reader (SpectraMax i3x). The CK activity was calculated according to the formula provided:
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