Cardiolipin assay kit

Myoblasts or myotubes were washed with PBS. After resuspension in cardiolipin assay buffer (1 mL per 100 mm dish), the cells were transferred to a new 1.5 mL E-tube and homogenized with a sonicator. After centrifugation at 10,000× g for 10 min at 4 °C, the supernatant was transferred to a new 1.5 mL E-tube. The extracted cardiolipin concentration was measured using a cardiolipin assay kit (Abcam) according to the manufacturer’s instructions; the concentration was calculated using the following formula: detection value (nmol)/sample volume (μL) × sample dilution factor of the standard curve.

The Cardiolipin Assay Kit from abcam (ab241036; Berlin, Germany) was used to analyze the levels of cardiolipin in SH-SY5Y mock and APPswedish transfected cells according to the manufacturer’s instructions.

In order to measure the cardiolipin levels via fluorescence, a Cardiolipin Assay Kit from Abcam (ab241036, Berlin, Germany) was utilized according to the manufacturer’s protocol.

Cardiolipin of isolated mitochondria was determined using a Cardiolipin Assay Kit (#ab241036, Abcam). Control and Acly knockdown BMDMs treated with IR-61 in the absence or with BMS-303141, then mitochondria were isolated and suspended in CL assay buffer. After centrifugation at 10,000g for 5 min, the supernatant was collected and mixed with CL probe. The fluorescence at Ex/Em 340/480 nm was recorded after incubating at room temperature for 10 min. Protein concentration was determined for normalization and the mitochondrial cardiolipin content was displayed as nmol mg⁻¹ protein.

The TA muscle was dissected from the mouse hindlimb and homogenized using the Minute™ Mitochondria Isolation Kit for Muscle Tissues (invent BIOTECHNOLOGIES). Isolated mitochondria were homogenized with a sonicator in cardiolipin assay buffer. Next, 10–20 μL of homogenized mitochondria was added to the cardiolipin assay plate, and the concentration of cardiolipin was measured with a cardiolipin assay kit (Abcam) according to the manufacturer’s instructions. The measured cardiolipin concentration was calculated using the following formula: detection value (nmol)/sample volume (μL) × sample dilution factor of the standard curve.

Citrate synthase activity and cardiolipin are reported as the best markers for mitochondrial content (Larsen et al., 2012 (link)). Citrate synthase activity was measured via production of coenzyme A (CoASH) from oxaloacetate. In the presence of 5,5′-dithio-bis- (2-nitobenzoic acid) (DTNB) and free sulfhydryl groups of coenzyme A react to form free 5-thio-2-nitrobenzoate anions which were measured at 412 nm (ε = 13.6 mM^–1cm^–1) using plate reader SpectraMax M5. The reaction mixture included 1 mM DNTB, 0.3 mM acetyl CoA, 1% sodium cholate, 200 µL of colon tissue homogenates. The reaction was initiated by adding 0.5 mM oxaloacetate. The rates were normalized to gram of tissue used to prepare the homogenate. The citrate synthase rates were used to normalize all mitochondrial rates to mitochondrial content. Cardiolipin was measured using the Abcam Cardiolipin Assay Kit (ab241036). Colon tissue homogenates were prepared and the assay was performed as outlined in the kit manual.