Gastrocnemius muscle was dissected and immediately frozen in liquid nitrogen, and then stored at −80 °C [36 (link)]. We used commercial assay kits (BC0515, BC3235, BC3245, BC0945; Solarbio, Beijing, China) to measure the enzyme activity of mitochondrial OXPHOS complexes of gastrocnemius muscle and DF-1 cells according to the manufacturer’s protocol. Complex I enzyme activity was determined by the change in absorbance of NADH as measured at 340 nm. Complex II enzyme activity was determined by the change in absorbance of DCIP as measured at 600 nm. The enzyme activity of complex III and complex IV was determined by the change in absorbance of reduced cytochrome c as measured at 550 nm. Absorbance was determined using a Fluorescence/Multi-Detection Microplate Reader (BioTek, Winooski, VT, USA) according to the manufacturer’s protocol. Data were normalized to the control group and expressed as a percentage of control levels.
Bc0945
Manufactured by Solarbio
Sourced in China
The BC0945 is a laboratory equipment product. It is designed for use in scientific research and analysis. The core function of this product is to provide a controlled environment or specialized tool for conducting experiments or measurements. No further details about the intended use or specific capabilities of this product are available.
Automatically generated - may contain errors
Lab products found in correlation
Bc3245, by Solarbio (6 mentions)
Bc0515, by Solarbio (5 mentions)
Bc3235, by Solarbio (5 mentions)
Bc1445, by Solarbio (4 mentions)
Bc0300, by Solarbio (3 mentions)
Bc3240, by Solarbio (3 mentions)
Bc3230, by Solarbio (2 mentions)
Bc0510, by Solarbio (2 mentions)
Fluorescence multi detection microplate reader, by Agilent Technologies (1 mentions)
Seahorse xf24 extracellular flux analyzer, by Agilent Technologies (1 mentions)
Varioskan lux microplate reader, by Thermo Fisher Scientific (1 mentions)
Mitochondria isolation kit, by Beyotime (1 mentions)
Atplite assay system, by PerkinElmer (1 mentions)
Infinite m200 pro plate reader, by Tecan (1 mentions)
Ca1410, by Solarbio (1 mentions)
Bc0630, by Solarbio (1 mentions)
Bc0950, by Solarbio (1 mentions)
M8650, by Solarbio (1 mentions)
Biotek synergy htx, by Agilent Technologies (1 mentions)
11 protocols using bc0945
1
Analyzing Mitochondrial OXPHOS Complexes
2
Mitochondrial function and glycolysis assessment
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The activity of mitochondrial respiratory complex I, II and IV, and ATP contents were measured by using commercial kits (BC0515, BC3235, BC0945, and BC0300, Solarbio, Beijing, China). The ECAR assay was conducted using a Seahorse XF-24 extracellular flux analyzer (Seahorse Bioscience, USA). Cells were seeded at a density of 8 × 105 cells per well. One day before measurement, the sensor cartridge was calibrated overnight. ECAR were measured sequentially before or after the addition of following reagents: oligomycin (1 μM), glucose (10 mM) and 2-DG (50 mM). Each measurement cycle included the following steps: 2 min mixture, 1 min waiting, and 3 min measurement. Following the last assay cycle, the plate was removed and cell counting was conducted. Total cell data was used to normalize the measured ECAR values.
3
Measuring Mitochondrial Complex IV Activity
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The COX activity was measured using a mitochondrial respiratory chain complex IV activity kit (BC0945; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). The difference in activity between the control and the bathocuproinedisulfonic acid (BCS) treatment groups was determined at 550 nm with a Varioskan LUX microplate reader (Thermo Fisher, Carlsbad, CA, USA) using the 1st and 15th min readings. Complex IV activity was normalized to the protein content.
4
Mitochondrial Function in Brain Samples
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Mitochondria were isolated from freshly acquired brain samples using a Mitochondria Isolation Kit (C3606, Beyotime). ATP content (BC0300, Solarbio) and mitochondrial ETC (I‐IV) complex (BC0515, BC3230, BC3240, BC0945; Solarbio) activities were assayed using the appropriate commercially available kits according to the manufacturer's instructions.
5
Enzymatic activity and mitochondrial function
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Complex (I, III, IV, and V) enzyme activity in cell extracts was measured by the single‐wavelength spectrophotometric assay using detection kits (cat. no. BC0510, BC3240, BC0945 and BC1445, Beijing Solarbio Science & Technology) according to the manufacturers’ protocols. Briefly, 5 × 106 cells were collected and homogenized in lysis buffer. After two steps of centrifugation (600 × g for 10 min and then 11, 000 × g for 15 min) at 4˚C, the precipitate was collected for ultrasonic crushing and then added into the corresponding substrate to start the enzymatic reaction. The absorbance values at different wavelengths (I: 340 nm; III: 550 nm; IV: 550 nm; V: 660 nm) of the metabolic product was detected using a spectrophotometer (Thermo Fisher Scientific Inc.).
The OCR was measured using a 782 Oxygen Meter (Strathkelvin Instruments, Motherwell, UK) according to the manufacturer's instructions. The amplified signal was recorded at sampling intervals of 0.5 s.
Mitochondrial ATP was measured as previously described [34 (link)]. Briefly, the abundance of ATP was determined using the ATPlite assay system (PerkinElmer Inc., Waltham, MA, USA). Basal luminescence was recorded before the use of luciferin, and then cells were perfused with 20 μmol/L luciferin to record mitochondrial luminescence [35 (link)].
The OCR was measured using a 782 Oxygen Meter (Strathkelvin Instruments, Motherwell, UK) according to the manufacturer's instructions. The amplified signal was recorded at sampling intervals of 0.5 s.
Mitochondrial ATP was measured as previously described [34 (link)]. Briefly, the abundance of ATP was determined using the ATPlite assay system (PerkinElmer Inc., Waltham, MA, USA). Basal luminescence was recorded before the use of luciferin, and then cells were perfused with 20 μmol/L luciferin to record mitochondrial luminescence [35 (link)].
6
Mitochondrial Complex III and IV Activity Assay
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Mitochondrial function was evaluated by using complex III detection kit (catalog:BC3240; Solarbio) and complex IV detection kit (catalog:BC0945；Solarbio). According to the manufacturer's protocol, A1 was determined as absorbance value of cell extract in 550 nm, and A2 was another measurement in 2 min later.36
7
Enzymatic activity of ETC complexes
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ETC complex activity detection kits (cat no. BC0515, BC3235, BC3245, BC0945 and BC1445; Beijing Solarbio Science & Technology, Co., Ltd.) were used to measure the enzymatic activity of ETC complexes I–V with an Infinite M200 PRO Plate Reader (Tecan Group, Ltd.) according to the manufacturer's protocol. The oxidation rate of NADH was recorded at 340 nm to assess the activity of ETC complex I. The rate of 2,6-dichloroindolephenol reduction was used to measure the activity of ETC complex II at 605 nm. The activity of ETC complex III was detected by measuring the increase rate of the light absorption of cytochrome c-reduced at 550 nm. The activity of ETC complex IV was monitored by measuring the rate of decrease in the absorbance of reduced cytochrome c at 550 nm. The activity of ETC complex V was quantified by calculating the rate of addition of inorganic phosphate (Pi) at 660 nm.
8
Mitochondrial Respiratory Chain Complex Assay
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Microglial mitochondrial respiratory chain complex I (BC0515, Solarbio), II (BC3235, Solarbio), III (BC3245, Solarbio), IV (BC0945, Solarbio) and ATP synthase (BC1445, Solarbio) activities were performed using corresponding assay kits according to the manufacturer’s instruction with slight adjustments. Briefly, at least 1.2 × 107 BV2 cells treated with or without FIR light at 0.13 mW/cm2 for 1 h were collected. After the medium were removed, 1 mL extract buffer was added to each sample, and homogenized on ice with a homogenizer. Subsequently, the homogenates were centrifuged at 500g for 5 min at 4 °C. The supernatants were transferred to another centrifugal tube, and centrifuged at 11,000g for 10 min at 4 °C. After the supernatants were removed, 400 μL extract buffer were added and these homogenates were then subjected to the ultrasonic lysis (power 52 W, ultrasonic 5 s, interval 10 s, for 15 times). The supernatant is used for mitochondrial respiratory chain complex activity determination. Finally, the enzymatic activity of these complexes was normalized to its protein content.
9
ICD Effects on Cell Metabolism
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Each well was seeded with 1×105 Cal-27 cells. After 24 h of incubation, different concentrations of ICD were added (0, 0.15, 0.30, 0.60, and 1.20 mM) for 24 h, 48 h, and 72 h, respectively. After the cells were harvested, the levels and activities of the MMP (M8650, Solarbio, Beijing, China), ATP (BC0300, Solarbio, Beijing, China), ROS (CA1410, Solarbio, Beijing, China), and mitochondrial complex enzymes I–IV (BC0630; BC0950; BC3245; BC0945, Solarbio, Beijing, China) were detected according to the manufacturer’s instructions. The changes in the MMP in each group were detected with a 490 nm excitation wave and a 530 nm irradiation wave. The ATP content of each group was detected at a 340 nm wavelength. ROS levels were measured at a 488 nm excitation wavelength and a 525 nm emission wavelength. In addition, the cellular ROS content was calculated using flow cytometry (CytoFlex, Indianapolis, IN, USA) after 24 h, 48 h, and 72 h of treatment with 0.60 mM ICD, respectively. The detection wavelength of complex enzyme I was 600 nm, while that of complex enzyme II was 600 nm, that of complex enzyme III was 550 nm, and that of complex enzyme IV was 550 nm. The activity of the respiratory electron transport chain complex enzymes I–IV was detected in each group.
10
Mitochondrial Respiratory Chain Assay
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We used a mitochondrial respiratory chain complex activity assay kit (BC0515, BC3235, BC3245, BC0945; Solarbio, Beijing, China) to detect the activities of CI, CII, CIII, and CIV in cells. A total of 5 × 106 cells were collected, homogenized, and centrifuged to collect the supernatants in a centrifuge tube. The sample was then centrifuged, and the resulting precipitate was resuspended in the extract and sonicated for disruption. The activity of the obtained liquid was determined according to specific protocols. The OD values of CI, CII, CIII, and CIV were measured by a multifunctional microplate detector (BioTek SynergyHTX, Agilent, Santa Clara, CA, USA) at 340 nm, 605 nm, 550 nm, and 550 nm.
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