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Succinate colorimetric assay kit

Manufactured by Merck Group
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The Succinate Colorimetric Assay Kit is a lab equipment product that enables the quantitative measurement of succinate levels in biological samples. It is based on a colorimetric detection method.

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13 protocols using succinate colorimetric assay kit

1

Quantifying TCA Cycle Metabolites

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To evaluate the TCA cycle in each group, the key metabolites, α-Ketoglutarate (α-KG) and succinate (Suc), were measured respectively. In our research, α-KG was detected with a-Ketoglutarate Assay Kit (MAK054, Sigma, USA), and Suc was measured using Succinate Colorimetric Assay Kit (MAK184, Sigma, USA) according to the manufacturer’s instructions.
Feng Y., Xu J., Shi M., Liu R., Zhao L., Chen X., Li M., Zhao Y., Chen J., Du W, & Liu P. (2022). COX7A1 enhances the sensitivity of human NSCLC cells to cystine deprivation-induced ferroptosis via regulating mitochondrial metabolism. Cell Death & Disease, 13(11), 988.
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2

Immunological and Metabolic Markers Quantification

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The blood samples collected from the experimental animal were allowed to stand and centrifuged at 1000× g for 15 min to obtain serum. The mouse IL-1β and IL-6 ELISA Kit (CUSABIO, Wuhan, China) were used to determine the levels of IL-1β and IL-6, respectively.
After treating bEnd.3 cells with the corresponding drugs, the supernatant was collected for the measurement of IL-1β (CUSABIO, Wuhan, China). In addition, the treated bEnd.3 cells were washed with PBS and collected with cell lysis buffer. After centrifuging at 12,000× g for 15 min at 4 °C, the supernatant was collected for the measurement of sAC, cAMP, and CaMKII according to the protocol of sAC ELISA Kit, cAMP ELISA Kit (Elabscience, Wuhan, China) and CaMKII ELISA Kit (Meimian, Yancheng, China). The quantification of succinate was measured by succinate colorimetric assay kit (Sigma, MAK184, St. Louis, MO, USA) The activity of CaN and SDH were detected according to the calcineurin assay kit (Jiancheng, A068-1-1, Nanjing, China) and succinate dehydrogenase assay kit (Jiancheng, A022-1-1, Nanjing, China).
Li Y.C., Li Y., Zhang Y.N., Zhao Q., Zhang P.L., Sun M.R., Liu B.L., Yang H, & Li P. (2022). Muscone and (+)-Borneol Cooperatively Strengthen CREB Induction of Claudin 5 in IL-1β-Induced Endothelium Injury. Antioxidants, 11(8), 1455.
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3

Mitochondrial Respiratory Complex Analysis

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Mitochondria were isolated from NRCMs using the Mitochondria Isolation Kit for Cultured Cells (ab110170, Abcam). The activities of complexes I and III in the mitochondrial ETC were assessed with the Complex I Enzyme Activity Assay Kit (ab109721, Abcam) and the Mitochondrial Complex III Activity Assay Kit (K520-100, Biovision) according to the manufacturer's instructions, respectively [44 (link)].
The contents of α-KG and Suc were measured using the α-Ketoglutarate Assay Kit (MAK054, Sigma) and the Succinate Colorimetric Assay Kit (MAK184, Sigma) in accordance with the manufacturer's instructions, respectively [44 (link)].
He H., Wang L., Qiao Y., Yang B., Yin D, & He M. (2021). Epigallocatechin-3-gallate pretreatment alleviates doxorubicin-induced ferroptosis and cardiotoxicity by upregulating AMPKα2 and activating adaptive autophagy. Redox Biology, 48, 102185.
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4

Metabolic Profiling of Cells under Hypoxia

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Cells were seeded into six-well plates at a density of 1 × 106 cells in 2 ml RPMI 1640 medium per well and cultured overnight. Cells were then treated with or without 10 μM KU60019 and maintained at 1% O2 for 10–12 h. Glucose consumption, lactate production, ATP production, citrate production, succinate production, fumarate production, and PFKP and CS activity were detected using glucose assay kit (Solarbio® BC2500), LA assay kit (Solarbio® BC2230), ATP content assay kit (Solarbio® BC0300), citric acid (CA) content assay (Solarbio® BC2150), micro mitochondrial citric acid content assay kit (Solarbio® BC2175), succinate colorimetric assay kit (Sigma® MAK184), pyruvate (PA) assay kit (Solarbio® BC2200), acetyl-CoA assay kit (Solarbio® BC0980), fumarate assay kit (Sigma® MAK060), PFKP test kit (Nanjing Jiancheng Bioengineering Institute A129), and citroyl synthetase kit (Nanjing Jiancheng Bioengineering Institute A108) according to instruction of the manufacturers, respectively. All experiments were performed at least three times and the data were normalized by the cell numbers or protein content.
Peng M., Yang D., Hou Y., Liu S., Zhao M., Qin Y., Chen R., Teng Y, & Liu M. (2019). RETRACTED ARTICLE: Intracellular citrate accumulation by oxidized ATM-mediated metabolism reprogramming via PFKP and CS enhances hypoxic breast cancer cell invasion and metastasis. Cell Death & Disease, 10(3), 228.
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5

Metabolic Profiling of Cells under Hypoxia

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Cells were seeded into six-well plates at a density of 1 × 106 cells in 2 ml RPMI 1640 medium per well and cultured overnight. Cells were then treated with or without 10 μM KU60019 and maintained at 1% O2 for 10–12 h. Glucose consumption, lactate production, ATP production, citrate production, succinate production, fumarate production, and PFKP and CS activity were detected using glucose assay kit (Solarbio® BC2500), LA assay kit (Solarbio® BC2230), ATP content assay kit (Solarbio® BC0300), citric acid (CA) content assay (Solarbio® BC2150), micro mitochondrial citric acid content assay kit (Solarbio® BC2175), succinate colorimetric assay kit (Sigma® MAK184), pyruvate (PA) assay kit (Solarbio® BC2200), acetyl-CoA assay kit (Solarbio® BC0980), fumarate assay kit (Sigma® MAK060), PFKP test kit (Nanjing Jiancheng Bioengineering Institute A129), and citroyl synthetase kit (Nanjing Jiancheng Bioengineering Institute A108) according to instruction of the manufacturers, respectively. All experiments were performed at least three times and the data were normalized by the cell numbers or protein content.
Peng M., Yang D., Hou Y., Liu S., Zhao M., Qin Y., Chen R., Teng Y, & Liu M. (2019). RETRACTED ARTICLE: Intracellular citrate accumulation by oxidized ATM-mediated metabolism reprogramming via PFKP and CS enhances hypoxic breast cancer cell invasion and metastasis. Cell Death & Disease, 10(3), 228.
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6

Quantifying SDH Activity and Succinate Levels

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SDH activity was measured using Succinate Dehydrogenase Assay Kit (Sigma, MAK197) according to manufacturer’s protocol. Briefly, at day 1 or 2 of differentiation in the presence of itaconate derivatives, cells were rinsed with PBS and lysed with SDH assay buffer. The whole cell lysate was freshly used for the measurement. SDH activity was calculated by measuring product absorbance at 600 nm every 5 min for 30 min at 25°C. SDH activity was normalized by protein contents in each sample measured by BCA assay. Succinate levels were determined using Succinate Colorimetric Assay Kit (Sigma, MAK184). After differentiation in the presence of itaconate derivatives, cells were rinsed with PBS and lysed with succinate assay buffer. The cell lysate was diluted 1:40 in assay buffer for analysis. After incubating samples with the reaction mix for 30 min at 37°C, the absorbance was measured at 450 nm. Concentration of succinate was normalized by protein contents in each sample measured by BCA assay.
Oh T.S., Hutchins D.C., Mainali R., Goslen K.H, & Quinn M.A. (2022). Itaconate and Its Derivatives Repress Early Myogenesis In Vitro and In Vivo. Frontiers in Immunology, 13, 748375.
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7

Quantification of Intracellular and Extracellular Succinate

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After stimulation, NRVMs (1 × 106) or cardiac tissue (10 mg) were rapidly homogenized on ice in 100 μL of succinate assay buffer. Then, the homogenate was centrifuged at 10,000 × g for 5 min to collect the supernatant. Intracellular succinate was detected using a Succinate Colorimetric Assay Kit (Sigma-Aldrich).
For measurement of extracellular succinate, NRVMs were treated with indicated agents, and then the conditional medium was collected for the assay according to the manufacturer’s instructions.
Lu Y.T., Li L.Z., Yang Y.L., Yin X., Liu Q., Zhang L., Liu K., Liu B., Li J, & Qi L.W. (2018). Succinate induces aberrant mitochondrial fission in cardiomyocytes through GPR91 signaling. Cell Death & Disease, 9(6), 672.
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8

Metabolic Profiling of Biological Samples

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The levels of glucose, lactate, and pyruvate were separately determined using the Glucose Assay Kit, Lactate Assay Kit, and Pyruvate Assay Kit (Jiancheng Bioengineering Institute, Nanjing, China). The levels of succinate and fumarate were separately measured by using the Succinate Colorimetric Assay Kit and the Fumarate Assay Kit (Sigma, St. Louis, MO). The levels of acetyl-CoA were tested using the Acetyl-Coenzyme assay kit and citrate levels were tested using the Citric Acid Assay Kit (Solarbio, China). The ATP products were measured using the ATP Assay Kit, and the mitochondrial membrane potential was determined using the JC-1 Dye (Beyotime, China). All these measurements follow their respective manufacturer’s protocols and were normalized by cell numbers or the quality of clinical sample. All experiments were performed at least three times.
Yang D., Peng M., Hou Y., Qin Y., Wan X., Zhu P., Liu S., Yang L., Zeng H., Jin T., Qiu Y., Li Q, & Liu M. (2020). Oxidized ATM promotes breast cancer stem cell enrichment through energy metabolism reprogram-mediated acetyl-CoA accumulation. Cell Death & Disease, 11(7), 508.
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9

Succinate Colorimetric Assay Protocol

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Succinate level was determined by Succinate Colorimetric Assay Kit (Sigma-Aldrich Inc., St Louis, MO, USA) according to the manufacturer's instructions. Briefly, tissues (50 mg) were rapidly homogenized on ice in 500 μL of ice-cold Succinate Assay Buffer and centrifuged at 10,000×g for 5 minutes to remove insoluble material. For cell cultures, the supernatants of cells (1×106 cells) were collected and diluted 1:1 with 0.5 M Tris-HCl (pH 8.0). Then, the samples were added into a 96-well plate in duplicate wells and mixed with the appropriate Reaction Mix. The resultant mixtures were further incubated at 37°C for 30 minutes. The succinate concentration was determined by the standard curve using spectroscopy at 450 nm wavelength, and each measurement was performed in triplicate.
Mu X., Zhao T., Xu C., Shi W., Geng B., Shen J., Zhang C., Pan J., Yang J., Hu S., Lv Y., Wen H, & You Q. (2017). Oncometabolite succinate promotes angiogenesis by upregulating VEGF expression through GPR91-mediated STAT3 and ERK activation. Oncotarget, 8(8), 13174-13185.
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10

Succinate Quantification in Adipocytes

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Differentiated adipocytes (106) stimulated with PA or TG (1 μM) for 8 h or 10 mg adipose tissue of HFD-fed mice were rapidly homogenized on ice in 100 μL of ice-cold succinate assay buffer. Centrifuge at 10,000 g for 5 min to collect the supernatant. The supernatant was measured by succinate colorimetric assay kit (Sigma, St Louis, USA) following the manufacturer's instructions.
Xiao N., Yang L.L., Yang Y.L., Liu L.W., Li J., Liu B., Liu K., Qi L.W, & Li P. (2017). Ginsenoside Rg5 Inhibits Succinate-Associated Lipolysis in Adipose Tissue and Prevents Muscle Insulin Resistance. Frontiers in Pharmacology, 8, 43.
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