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Study Materials

Protocols, software, and reference materials used in the laboratory — shared in the interest of reproducible bioenergetic research.

The TEMPO@UVA Laboratory is committed to methodological transparency. The materials below document the measurement protocols, analysis pipelines, and reference standards we use day to day. Wherever licensing and data-use agreements permit, we make these available to collaborators, trainees, and the broader bioenergetics community.

Availability note: several items are being prepared for public release as the laboratory establishes its infrastructure at UVA. If you need a protocol or analysis tool before it is posted, email bi9n@virginia.edu and we will share the current version directly.

Measurement protocols

In vivo — near-infrared spectroscopy

Protocol	Device	Primary outcome	Notes
Mito-6 paradigm	Artinis PortaMon, Train.Red	Rate constant of muscle reoxygenation (k, min⁻¹)	Six repeated arterial occlusions following brief exercise; mono-exponential fit to recovery kinetics.
Resting mV̇O₂	Artinis PortaMon, Train.Red	Resting muscle oxygen consumption	Single sustained occlusion; slope of desaturation during ischemia.

EX VIVO — HIGH-RESOLUTION FLUORO-RESPIROMETRY

Protocol	Preparation	MEASUREMENT
Substrate Uncoupler Inhibitor Titration (SUIT) Protocols	Permeabilized fibers; isolated mitochondria tissue homogenates	Oxygen Flux (JO2)
	Intact cells; Permeabilized cells	Oxygen Flow (IO2)
Amplex Red Assay	Isolated mitochondria; permeabilized cells	Simultaneous JO2 and JH2O2 redox balance measurements
NADH Fluorometry (NextGen O2k)	Isolated mitochondria	Simultaneous JO2 and NAD+/NADH redox balance measurements
Immune Cell Bioenergetics	PBMC, T-cells, NK-cells	Intact and permeabilized cell respiration

Whole-body physiological assessment

Cardiopulmonary Exercise Testing (CPET) — graded exercise testing protocols for measuring V̇O₂peak, ventilatory thresholds, and substrate oxidation via indirect calorimetry.

Isokinetic Dynamometry — peak torque, work, and fatigability indices across a standardized velocity spectrum.

Body Composition Assessment — total and regional lean mass, fat mass, appendicular lean mass index, bone mineral density, and trabecular bone score by DXA under Certified Clinical Densitometrist supervision. Additional expertise in CT, MRI, Ultrasound, BIS, and 3D Body Scans. D3-Creatine dilutions methods in development.

Stable-Isotope Tracers — to measure protein synthesis and substrate kinetics.

Reference standards & terminology

The laboratory follows internationally harmonized conventions for reporting mitochondrial respiratory states and rates. Trainees are expected to become familiar with these before generating or interpreting respirometry data.

MitoEAGLE consensus — respiratory states and rates

Gnaiger E and the MitoEAGLE consortium (including Dr. Irving). The reference framework for naming and reporting mitochondrial respiratory measurements.

Citation

Resolving the Rotenone Paradox

Chicco et al., MitoFit Preprints 2022. Methodological treatment of multi-substrate SUIT protocol complexity — required reading for respirometry trainees.

Citation

Precision exercise medicine — response variability

Ross et al., Br J Sports Med 2019. Framework for interpreting individual variability in exercise training response.

Citation

For laboratory trainees

New members of the laboratory should complete the following before independent data collection.

Interested in joining? Prospective undergraduate, graduate, and postdoctoral trainees should visit the Get Involved page for current openings and application guidance.

UVA human-subjects research (CITI) training and IRB protocol enrollment

Laboratory safety, bloodborne pathogen, and biosafety training as applicable to your project

Supervised practice on the relevant instrument until measurement reproducibility meets lab criteria

Review of the MitoEAGLE terminology framework and the lab's SUIT protocol documentation