The sensitivity of mPTP to Ca2+ could reflect the opening of mPTP [9 (link)]. The mitochondria of SA-treated cardiomyocytes were extracted from the cell lysates using Cell Mitochondria Isolation Kit (Beyotime) following to the manufacturer’s recommendations. The reaction of mPTP to Ca2+ was detected using Purified Mitochondrial Membrane Pore Channel Colorimetric Assay kit (GENMED, Shanghai, China) following to the manufacturer’s recommendations.
Purified mitochondrial membrane pore channel colorimetric assay kit
Manufactured by Genmed Scientifics
Sourced in China
The Purified Mitochondrial Membrane Pore Channel Colorimetric Assay kit is a laboratory tool used to measure the activity of mitochondrial membrane pore channels. The kit provides a colorimetric-based method for quantifying the opening and closing of these pores in a sample. The core function of this product is to enable researchers to study mitochondrial membrane pore channel dynamics.
Automatically generated - may contain errors
Lab products found in correlation
Ultramicro microporous plate spectrophotometer, by Agilent Technologies (3 mentions)
Cell mitochondria isolation kit, by Beyotime (2 mentions)
Tissue mitochondria isolation kit, by Beyotime (1 mentions)
Calcein am, by Thermo Fisher Scientific (1 mentions)
Tissue cell mitochondria isolation kit, by Beyotime (1 mentions)
Cocl2, by Merck Group (1 mentions)
6 protocols using purified mitochondrial membrane pore channel colorimetric assay kit
1
Investigating Mitochondrial Permeability Transition Pore
2
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.
3
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)).
4
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)].
5
Mitochondrial Membrane Pore Assay
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The mitochondria derived from rat myocardium and/or from H9c2 cells were isolated by differential centrifugation using a Tissue/Cell Mitochondria Isolation Kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. Fresh mitochondria were used for the measurement of mPTP opening, while the mitochondria-free cytoplasmic protein extract was used for the measurement of cytochrome c expression.
mPTP opening was measured using a Purified Mitochondrial Membrane Pore Channel Colorimetric Assay kit (Genmed). mPTP opening was induced by 200 µM CaCl2 and presented as mitochondrial swelling, which results in a reduction of the absorbance at 520 nm (A520). The changes in A520 at various time points were measured for each sample. The value at −1 min was normalized to the value at 10 min and was used for statistical analysis.
An additional immunofluorescence method was used to measure mPTP opening in H9c2 cells. Briefly, cells were washed with PBS and subsequently stained with 1 µmol/l calcein-AM (Invitrogen; Thermo Fisher Scientific, Inc.) in the presence of 8 mmol/l CoCl2 (Sigma-Aldrich; Merck KGaA) at room temperature for 20 min in the dark. CoCl2 was added to quench the cytoplasmic signal so that only fluorescence in the mitochondria was captured. A change in fluorescence intensity corresponded to the level of mPTP opening.
mPTP opening was measured using a Purified Mitochondrial Membrane Pore Channel Colorimetric Assay kit (Genmed). mPTP opening was induced by 200 µM CaCl2 and presented as mitochondrial swelling, which results in a reduction of the absorbance at 520 nm (A520). The changes in A520 at various time points were measured for each sample. The value at −1 min was normalized to the value at 10 min and was used for statistical analysis.
An additional immunofluorescence method was used to measure mPTP opening in H9c2 cells. Briefly, cells were washed with PBS and subsequently stained with 1 µmol/l calcein-AM (Invitrogen; Thermo Fisher Scientific, Inc.) in the presence of 8 mmol/l CoCl2 (Sigma-Aldrich; Merck KGaA) at room temperature for 20 min in the dark. CoCl2 was added to quench the cytoplasmic signal so that only fluorescence in the mitochondria was captured. A change in fluorescence intensity corresponded to the level of mPTP opening.
6
Calcium Regulation of Mitochondrial Pore
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The sensitivity of mPTP to calcium ion (Ca2+) could reflect the opening of mPTP.17 To be specific, the mitochondria in transfected cardiomyocytes were extracted from the cell lysates using Cell Mitochondria Isolation Kit (Beyotime) following to the manufacturer's recommendations. The reaction of mPTP to Ca2+ was detected using Purified Mitochondrial Membrane Pore Channel Colorimetric Assay kit (GENMED) following to the manufacturer's recommendations.
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