Changes in redox status can have a major impact on cellular and physiologic processes and are believed to underlie many functional decrements associated with the aging process. Cellular energy production and survival depends upon a series of oxidation/reduction reactions. These reactions can give rise to free radical and pro-oxidant species that can act as regulatory molecules and induce oxidative damage. Thus, the redox potential of a cell is a reflection of multiple interacting molecules and biological processes that influence both oxidant production and removal and ultimately cellular homeostasis.

Oxidative Damage: Oxidative damage in lipids, DNA, and proteins will be measured by the levels of F2-isoprostanes (lipid peroxidation) and protein carbonyls.

The Core provides investigators with services to measure mitochondrial function (oxidant generation, ATP production, respiration) as well as energy charge (ATP, ADP, and AMP) in fresh tissue samples or isolated mitochondria. This includes in vitro analyses of mitochondrial function in isolated mitochondria, respirometry analysis in fresh tissue using the Oroborus respirometer, as well as measurement of mitochondrial function in cells using the Seahorse XF24 Extracellular Flux Analyzer. While energy status can be measured on flash frozen tissues/cells, assays of mitochondria function must be performed with fresh tissue/cells or freshly-isolated mitochondria.

The design of the services for Single-Cell RNA Sequencing (scRNA-Seq) is in three tiers so that investigators can tailor their studies to the number of cells that need to be analyzed, the depth of transcriptome coverage, and their available resources. At the broadest reach, 10X Genomics Chromium instrumentation services enable analysis of 1,000s of cells per sample with 100s to low 1,000s of mRNAs per cell. However, these are significant studies and cell preparation approaches must be optimized for each project. A less expensive, lower cell number (100s) approach such as Illumina/BioRad ddSeq is an attractive alternative for initial work.

nicotinamide adenine dinucleotides (NAD+, NADH, & NADP+, NADPH)

Methodology:  LC/MS

Cost: $50/sample

TCA intermediates

Methodology:  LC/MS

Cost: $50/sample

Both nicotinamide adenine dinucleotides and TCA

Methodology:  LC/MS

Cost: $60/sample

Other metabolites

Methodology:  LC/MS

Cost: available upon request

Reduced and oxidized glutathione (GSH and GSSG)

Methodology:  HPLC with electrochemical detection

Cost: $50/sample

F2-isoprostanes (lipid peroxidation)

Methodology:  GC/MS

Cost: $100/sample

Protein oxidation

Methodology:  Fluorescent derivatization and SDS PAGE

Cost: $50/sample

Protein oxidation

Methodology:  Fluorescent derivatization and 2D PAGE

Cost: $60/sample

Mitochondrial function

Methodology:  Seahorse XFe24 or XFe96 - Mito Stress Test; includes normalization with Cytation 5 Imager

Cost: $200/plate

Mitochondrial functional analysis on frozen tissue

Methodology:  Spectrophotometric based assays of NADH oxidase, complex I, other ETC

Cost: $25/sample

ROS/RNS

Methodology:  EPR Spin Tapping

Cost: available upon request