For mitochondrial substrate functional assessment, we used a previously described protocol optimised for hPSC-CMs. 30 Briefly, MitoPlates (Biolog) were prepared with 30 mL assay buffer mix containing 50 mg/mL Saponin (Sigma-Aldrich) and incubated at 37 C for 1 h. Cells were dissociated, resuspended in 1x MAS Buffer (Biolog) at a concertation of 2x106 cell/mL, of which 30 mL added into each well of the MitoPlate. The MitoPlate was read for 6 h with 5-min intervals at OD590 using a OmniLog plate reader. OmniLog peak value change was calculated via provided software Data Analysis 1.7.
Mitoplate
Manufactured by Biolog
Sourced in United States
The MitoPlate is a specialized laboratory equipment designed for the analysis of mitochondrial function. It provides a standardized platform for the measurement of various mitochondrial parameters, including oxygen consumption, ATP production, and metabolic activity. The MitoPlate enables researchers to study the role of mitochondria in cellular processes and its potential implications in various fields of study.
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5 protocols using mitoplate
1
Mitochondrial Substrate Function Assay
2
Mitochondrial Respiratory Profiling of CD117+ Cells
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MitoPlates were prepared following manufacturer’s instructions; 30 μL assay mix containing 50 μg/mL saponin (47036-50G-F; MilliporeSigma) was pipetted into the wells and incubated at 37°C for 1 hour. During this incubation, CD117+ cells were harvested as described earlier, filtered by using a 40 μm nylon filter, and counted. Cells were pelleted, resuspended in 1x MAS Buffer (72303; Biolog, Inc., Hayward, CA), creating a final concentration of 2.5 × 106 cells/mL; 30 μL of cell resuspension was then pipetted into each well of the MitoPlate (14105; Biolog, Inc.). The MitoPlate was read for 6 hours with 5-minute intervals at an optical density (OD) of 590 nm using the OmniLog plate reader. The initial and final OD readings were separated and organized per well. Data were restructured to fit correct MitoPlate format, the final OD reading was subtracted against the initial OD, and any negative results were set to 0. The triplicate no-substrate control wells were averaged; each individual calculated OD value was subtracted from this value and averaged, creating the average aggregate minus no substrate table. The negatives were removed from this table and the data reorganized to match each substrate. To create the heatmaps, final data were normalized to young control and highlighted using 3-color conditional formatting.
3
Mitochondrial Function Assay in Lymphoma Cells
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TCA function was identified in ATM−/− DLBCL cells using MitoPlate S-1 assay (BiOLOG, CA, USA) as per protocols described in38 (link), which were modified to use in lymphoma cells. BiOLOG assay is a colorimetric-based method, which utilizes redox dye and provides a high-resolution approach to measure mitochondrial function. Briefly, cells were permeabilized with permeabilizing buffer, followed by, subsequent addition of dye mix from (BiOLOG, CA, USA). Formation of air bubbles upon dye mix addition was carefully avoided. We used 50,000 cells/well for BiOLOG MitoPlate assays. Beginning after a one-hour incubation period, color measurement was captured every 15 min for two hours using a plate reader (Synergy H1M) (BioTek, Winooski, VT, USA) that allowed kinetic reading at 590. Data was plotted as percentage utilization of substrate in ATM−/− cells compared with cells expressing wild-type ATM.
4
Mitochondrial Metabolic Profiling and Iron Analysis
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Mitochondrial substrate utilization was conducted according to the manufacturer’s instructions (Biolog, Hayward, CA, USA) in which viable cell number was first determined by trypan blue exclusion, and plated in Biolog Mitoplates. A 96 well plate reader was used in kinetic mode at OD590 over 4 h and the relative amount of substrate utilization was determined by the change in absorbance over this time period. To measure total non-heme iron, cells at 70% confluency were utilized. Media was collected 1:1 with 3 N HCl/10% trichloroacetic acid (TCA); cells were washed with PBS, scraped into 1.0 mL 3 N HCl/10% TCA and stored at room temperature. For analysis, after hydrolysis at 70 °C for 24 h precipitating heme in 10% TCA, iron (non-heme) concentrations in the supernatant were determined using ICPOES (Model Optima 4300D, Perkin Elmer, Norwalk, CT, USA) operated at a wavelength of 238.204 nm. The in-gel aconitase activity assay was conducted as described [42 (link)], loading 70 μg protein per well. Succinate dehydrogenase activity was determined according to the manufacturer’s instructions (Abcam, ab228560).
5
Mitochondrial Metabolism in Rat Hippocampal Neurons
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Primary rat hippocampal neuron cultures were plated in 96-well plates at 27,000 cells/cm2 for 48 h. Cells were then exposed to BDE-47, BDE49, or vehicle (1:1000) in the absence or presence of T3 in combination with a mitochondrial substrate library, MitoPlate-S (Biolog, Inc., Hayward, CA, USA). Mitochondrial substrate metabolism was characterized according to the manufacturer’s protocol. Kinetics was recorded on the H1 hybrid microplate reader at a wavelength of 590 nm.
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