Laboratory Research

A shift between respiration and glycolysis is observed in several pathological states including cancer, obesity, diabetes, and mitochondrial, cardiovascular, and neurodegenerative diseases. Cellular bioenergetics also play a role in aging, immune response, hypoxia and drug toxicity.

Our core provides comprehensive services for analysis of metabolism and mitochondrial function in the research setting. Instrumentation includes a Seahorse XFe96 Extracellular Flux Analyzer for simultaneous measurement of Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) in order to interrogate changes in oxidative phosphorylation and glycolysis in cultured cells, isolated mitochondria, islets or small tissue samples. This bioenergetic analysis allows testing multiple conditions per assay well and direct, simultaneous comparisons across wells, enabling faster throughput with smaller samples compared with traditional respirometry methods.

The Seahorse Flux Analyzer also measures bioenergetic phenotyping and analysis for all sorts of samples.

Facilites and Equipment

Location: DA- 605, 330 Brookline Ave, Boston

Equipment: XFe96 Extracellular Flux Analyzer, Prep Station

The maximum amount of time per run is 3 hours (including cartridge calibration).

It is the responsibility of the user to ensure that the sensor cartridge be hydrated overnight prior to use. The Core has a non-CO₂ incubator which can be used for preparation of the cartridge.

Appointments for use of this instrument can be made by.

Training

Training is required before use of this instrument.

Individual consultation and training may be scheduled by contacting the Core.

First time users should contact at least 2 weeks before running an experiment.

Links

• http://www.agilent.com/cs/agilent/en-us/promotions/xftechnologyoverview
• Learning to use the Seahorse for metabolic analysis

http://www.agilent.com/en-us/products/cell-analysis-(seahorse)/seahorse-analyzers/seahorse-xfe96-analyzer/basic-procedures-to-run-an-xfe96-assay

• Scientific applications webinars on demand

http://www.agilent.com/en-us/training-events/eseminars

• Manuals and Literature

Services

1. Basic Assay
   – core provides assay plate and assay cartridge
   – user provides samples in the assay plate, assay medium, and injection reagents
2. Seahorse XF Glycolytic Rate Assay
3. Seahorse XF Glycolysis Stress Test
   – core provides assay plate, assay cartridge, Seahorse medium with supplements, and Seahorse reagents
   – user provides samples in the assay plate
4. Seahorse XF Cell Mito Stress Test
   – core provides assay plate, assay cartridge, Seahorse medium with supplements, and reagents
   – user provides samples in the assay plate
5. Seahorse XF Mito Fuel Flex Test
6. Seahorse XF Cell Energy Phenotype Test

All services include use of the Seahorse Prep Station liquid handling system and non-CO₂ incubator, cartridge hydration on the day prior to the assay, and technical assistance. Users are responsible for preparing media and injection reagents, performing media exchanges, loading the assay cartridges, running the assay, and analyzing the data. All new users require training.

Cost

Basic Procedures to Run an XF^e96 Assay

Planning Your XFe96 Assay

Materials Required to Run an XFe96 Assay
A list of the materials required or recommended to run a successful XFe96 assay.

Procedures Required the Day Before the XFe96 Assay

Seeding Cells in XFe96 Cell Culture Microplates
This basic procedure describes recommendations for seeding adherent cell types for XF assay in the XFe96 Analyzer.

Hydrating the Sensor Cartridge for the XFe96 Analyzer
This basic procedure describes the recommended procedure and conditions for hydrating an XFe96 Sensor Cartridge before running the XF assay.

Procedures Required the Day of the XFe96 Assay

Preparing Assay Media for Use in XF Assays
This basic procedure details the preparation of assay media for use with the XF Cell Mito Stress Test Kit, and XF Glycolysis Stress Test.

Washing Cells in XFe96 Cell Culture Microplates
This procedure describes the procedure for replacing growth medium with assay medium for adherent cells grown in XF96 cell culture microplates prior to being assayed using an XFe96 Analyzer.

Loading the XFe96/XF96 Sensor Cartridge with Compounds
This basic procedure provides cartridge loading instructions for XF assays on the XFe96 Analyzer.

Running the XF Assay

Cell Density and Oligomycin Optimization with the XF Glycolysis Stress Test
This basic procedure details the four different cell densities, and four different concentrations of oligomycin required to determine the optimal cell density and oligomycin concentration for use in an XF assay
FCCP Optimization with the XF Cell Mito Stress Test
This basic procedure details the six different concentrations of FCCP required to determine the optimal FCCP concentration for use an XF assay.

Seahorse XF Glycolysis Stress Test

• Provides a robust test for analyzing the key parameters of glycolytic function
• Measure: glycolytic capacity, glycolytic reserve, and non-glycolytic acidification.
• Kit provides quality-controlled, pre-measured reagents and a standard method for conducting a Seahorse XF Glycolysis Stress Test
• Seahorse XF Report Generator automatically calculates the key parameters of your samples, simplifying data analysis
• Seahorse XF Stress Test Report Generator
• Let’s talk about Glycolytic Activity

Mito Stress Assay

• Provides a robust test for analyzing the key parameters of mitochondrial function
• Measure: Basal respiration, ATP-linked respiration, H+ (Proton) Leak, Maximal Respiration, Spare Respiratory Capacity, and Non-mitochondrial respiration.
• Kit provides quality-controlled, pre-measured reagents and a standard method for conducting a Seahorse XF Cell Mito Stress Test
• Seahorse XF Report Generator automatically calculates the key parameters of your samples, simplifying data analysis
• Seahorse XF Stress Test Report Generators
• Let’s talk about Mitochondrial Respiration

Seahorse XF Cell Energy Phenotype Test

• Provides a routine test for monitoring the Energy Phenotype and Metabolic Potential of live cells
• A simple, sensitive method for comparing metabolic phenotypes between large groups of samples
• Kit provides quality-controlled, pre-measured reagents and a standard method for conducting a Seahorse XF Cell Energy Phenotype Test
• Seahorse XF Report Generator automatically determines the Energy Phenotype and Metabolic Potential of your samples, simplifying data analysis
• Seahorse XF Cell Energy Phenotype Test Report Generator
• Let’s Talk about Seahorse XF Cell Energy Phenotype Test

Seahorse XF Mito Fuel Flex Test

• Provides a single test for analyzing three fuel oxidation pathways: Glucose, Glutamine, and/or Fatty Acid
• Measure: fuel dependency and fuel pathway flexibility
• Kit provides quality-controlled, pre-measured reagents and a standard method for conducting a Seahorse XF Mito Fuel Flex Test
• Seahorse XF Report Generator automatically determines the key parameters of your samples, simplifying data analysis
• Seahorse XF Mito Fuel Flex Test Report Generator
• Let’s talk about Mitochondrial Fuel Flexibility

MESO QuickPlex SQ 120 is a compact, affordable multiplexing imager with exceptional performance offering affordable access to high performance, electrochemiluminescence immunoassays. This instrument uses Meso Scale Discovery (MSD)’s proprietary MULTI-ARRAY technology to provide unparalleled sensitivity and dynamic range, simple protocols, rapid and continuous reads, and fast, well-organized results via DISCOVERY WORKBENCH assay analysis software. They require no customer calibration or maintenance, no complicated fluidics, and no between-read cleaning. You can multiplex biomarker and cytokine assays, test for immunogenicity or toxicity, or develop your own unique assays. The technology makes the process fast, simple, low sample volume requirements, and minimal consumption of reagents.

Meso Scale Discovery immunoassay kits provide highly sensitive, precise, and accurate results across a wide dynamic range with fast, efficient processing. For all available assays, please check out the following website.

https://www.mesoscale.com/en/applications

Electrochemiluminescent

Electrochemiluminescent labels generate light when stimulated by electricity in the appropriate chemical environment. This reaction is incorporated into our immunoassays to provide the light signal used to measure important proteins and other biomedical molecules.

How does electrochemiluminescence work?

1. High binding carbon electrodes in the bottom of MULTI-ARRAY and MULTI-SPOT microplates allow for easy attachment of biological reagents (10X greater binding capacity than polystyrene).
2. MSD assays use electrochemiluminescent labels that are conjugated to detection antibodies. The labels are called SULFO-TAG, and allow for ultra-sensitive detection.
3. Electricity is applied to the plate electrodes by an MSD instrument leading to light emission by SULFO-TAG labels. Light intensity is then measured to quantify analytes in the sample.

Advantages of Electrochemiluminescence:

• High sensitivity: Multiple excitation cycles can amplify signals to enhance light levels.
• Broad dynamic range: The wide dynamic range of our detection systems means high and low expression levels can be measured without multiple sample dilutions.
• Low background: The stimulation method (electricity) is decoupled from the signal (light) allowing only labels near the electrode surface to be detected.
• Easy to use: Immunoassay method similar to conventional ELISA, but quicker.
• Great flexibility: Labels are stable, non-radioactive, and conveniently conjugated to biological molecules.
• Unsurpassed performance and quality: Electrochemiluminescence is a highly successful detection system that achieves clinical quality data in a variety of sample types, including cell supernatant, serum, plasma, and whole blood.

Mitochondrial Bioanalytical Services

• mtDNA Damage Analysis
• Cytochrome c Assay
• Mitochondrial PDH activity and quantity Assays
• Mitochondrial Citrate Synthase Assay

Cost