Cell viability was determined using the Cell Counting Kit-8 (CCK-8) assay (Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. Absorbance was detected at 450 nm using the Ultramicro Microporous Plate Spectrophotometer (BioTek, Winooski, VT).
Ultramicro microporous plate spectrophotometer
Manufactured by Agilent Technologies
Sourced in United States
The Ultramicro microporous plate spectrophotometer is a laboratory instrument designed for the measurement of light absorbance in small sample volumes. It is capable of performing spectrophotometric analysis on samples contained in microplate formats.
Automatically generated - may contain errors
Lab products found in correlation
Purified mitochondrial membrane pore channel colorimetric assay kit, by Genmed Scientifics (3 mentions)
Ldh assay kit, by Nanjing Jiancheng (2 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
Tissue mitochondria isolation kit, by Beyotime (1 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (1 mentions)
Ck mb assay kit, by Nanjing Jiancheng (1 mentions)
Cck 8 assay, by Dojindo Laboratories (1 mentions)
Cell counting kit 8 (cck8), by Biosharp (1 mentions)
11 protocols using ultramicro microporous plate spectrophotometer
1
Cell Viability Assay: CCK-8 Protocol
2
Measuring Cardiac Enzyme Levels
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LDH and CK-MB are released from cardiomyocytes into the coronary effluent during perfusion (29 (link)). The activities of LDH and CK-MB in the coronary effluent were determined using the LDH and CK-MB assay kits (Nanjing Jiancheng Bioengineering Institute; cat. nos. A020-1 and E006) according to the manufacturer's protocols. The absorbance value was detected at 450 nm by ultramicro microporous plate spectrophotometer (BioTek Instruments, Inc.).
3
Mitochondrial Permeability Transition Pore Assay
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The mitochondria were isolated from heart tissues using Tissue Mitochondria Isolation kit (Beyotime Institute of Biotechnology) according to the manufacturer’s protocol. The reaction of MPTP to calcium was determined using Purified Mitochondrial Membrane Pore Channel Colorimetric Assay kit (GENMED, Shanghai, China) according to the manufacturer’s protocol.22 (link) Specifically, MPTP opening was induced by CaCl2. The value of optical density (OD) was read at 520 nm from 0 to 10 mins by an ultramicro microporous plate spectrophotometer (Biotek, USA). The decrease in OD reflected the extent of MPTP opening.
4
Measuring H9c2 Cell LDH Activity
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LDH activity of the H9c2 cells was determined by LDH assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s protocols at 450 nm by ultramicro microporous plate spectrophotometer (Biotek, USA).
5
Mitochondrial Permeability Transition Pore Assay
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The opening of the mPTP was measured by detecting the A540 absorbance of mitochondria exposed to 250 μM CaCl2 (“+”: treatment with Ca+; “–”: treatment without Ca+). Purified Mitochondrial Membrane Pore Channel Colorimetric Assay kit (GENMED, Shanghai, China) was used for detection; 200 μmol/L CaCl2 was used to induce mPTP opening. An ultramicro microporous plate spectrophotometer (Biotek, USA) was used to read the value of optical density (OD) from 0 to 10 min at 520 nm. The OD decrease reflected the mPTP opening. The OD value noted at the onset of the experiment (0 min) represented the minimum optical density (min OD); the OD value noted at the end of the experiment (10 min) represented the maximum optical density (min OD). The min/max OD was negatively associated with the extent of MPTP opening (34 (link), 47 (link)).
6
Cell Viability Assessment by CCK-8 Assay
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Cell viability was determined by CCK-8 assay using the Cell Counting Kit-8 (Dojindo Molecular Technologies, Kumamoto, Japan) according to the manufacturer’s protocol. The absorbance value was detected at a wavelength of 450 nm by ultramicro microporous plate spectrophotometer (Biotek Instruments, Inc., Winooski, UT, USA).
7
Colorimetric Assay for mPTP Opening
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Opening of the mPTP was measured by detecting absorbance at 540 nm of mitochondria exposed to 250 μM CaCl2; “-”: treatment without Ca+; “+”: treatment with Ca+. The Purified Mitochondrial Membrane Pore Channel Colorimetric Assay kit (GENMED, Shanghai, China) was used according to the manufacturer’s protocol. CaCl2 (200 μmol/L) was used to induce opening of the mPTP. The optical density (OD) value from 0 to 10 min at 520 nm was read from an ultra-micro microporous plate spectrophotometer (Biotek, Winooski, VT, USA). A decrease in the OD value reflects opening of the mPTP. The OD value recorded at the onset of the experiment (0 min) represents minimum optical density (min OD); the OD value recorded at the end of the experiment (10 min) represents maximum optical density (min OD). Min/max OD is negatively associated with the extent of MPTP opening [24 , 25 (link)].
8
Cardiomyocyte Enzyme Release Assay
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Lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) released from cardiomyocytes into the culture medium were determined using the LDH Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and CK-MB Assay Kit (Nanjing Jiancheng Bioengineering Institute), respectively, according to the manufacturer’s protocols. Absorbance was detected at 450 nm using the Ultramicro Microporous Plate Spectrophotometer (BioTek).
9
Investigating lncRNA GAS5 in MIRI
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To investigate the effect of lncRNA GAS5 on cell viability in MIRI, a CCK-8 assay was used. The H9c2 cells (3×103 cells/well) were cultured in 96-well plates according to the experimental groups, and then cell viability was determined using a CCK-8 assay (Dojindo Molecular Technologies, Inc.), according to the manufacturer's protocol. Subsequent to treating the cells according to the predetermined experimental groups, 10 µl CCK-8 solution was added to each well and incubated for 1 h at 37°C in a 5% CO2 incubator. The absorbance value was detected at a wavelength of 450 nm by an ultra-micro microporous plate spectrophotometer (BioTek Instruments, Inc.).
10
Evaluating Myocardial Cell Injury Using LDH and CK-MB
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The H9c2 cells (3×103 cells/well) were cultured in 96-well culture plates to detect myocardial enzyme markers. Lactate dehydrogenase (LDH) and creatine kinase isoenzyme (CK-MB) may be released into the culture medium following myocardial cell injury and death, and their levels may be tested to indicate the degree of damage to cardiomyocytes (31 (link)). LDH was determined using an LDH assay kit (Shenyang Wan Biotechnology Co., Ltd.) according to the manufacturer's protocol. The levels of CK-MB in the culture medium were measured using a CK-MB enzyme-linked immunosorbent assay kit (cat. no. MB-6930A; Jiangsu MB Biotechnology Co., Ltd.) according to the manufacturer's protocol. The absorbance value was detected at a wavelength of 450 nm by an ultra-micro microporous plate spectrophotometer (BioTek Instruments, Inc.).
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