Use of Electron Paramagnetic Resonance in Biological Samples at Ambient Temperature and 77 K

Summary

Electron paramagnetic resonance (EPR) spectroscopy is an unambiguous method to measure free radicals. The use of selective spin probes allows for detection of free radicals in different cellular compartments. We present a practical, efficient method to collect biological samples that facilitate treating, storing, and transferring samples for EPR measurements.

Abstract

The accurate and specific detection of reactive oxygen species (ROS) in different cellular and tissue compartments is essential to the study of redox-regulated signaling in biological settings. Electron paramagnetic resonance spectroscopy (EPR) is the only direct method to assess free radicals unambiguously. Its advantage is that it detects physiologic levels of specific species with a high specificity, but it does require specialized technology, careful sample preparation, and appropriate controls to ensure accurate interpretation of the data. Cyclic hydroxylamine spin probes react selectively with superoxide or other radicals to generate a nitroxide signal that can be quantified by EPR spectroscopy. Cell-permeable spin probes and spin probes designed to accumulate rapidly in the mitochondria allow for the determination of superoxide concentration in different cellular compartments.

In cultured cells, the use of cell permeable 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) along with and without cell-impermeable superoxide dismutase (SOD) pretreatment, or use of cell-permeable PEG-SOD, allows for the differentiation of extracellular from cytosolic superoxide. The mitochondrial 1-hydroxy-4-[2-triphenylphosphonio)-acetamido]-2,2,6,6-tetramethyl-piperidine,1-hydroxy-2,2,6,6-tetramethyl-4-[2-(triphenylphosphonio)acetamido] piperidinium dichloride (mito-TEMPO-H) allows for measurement of mitochondrial ROS (predominantly superoxide).

Spin probes and EPR spectroscopy can also be applied to in vivo models. Superoxide can be detected in extracellular fluids such as blood and alveolar fluid, as well as tissues such as lung tissue. Several methods are presented to process and store tissue for EPR measurements and deliver intravenous 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CPH) spin probe in vivo. While measurements can be performed at room temperature, samples obtained from in vitro and in vivo models can also be stored at -80 °C and analyzed by EPR at 77 K. The samples can be stored in specialized tubing stable at -80 °C and run at 77 K to enable a practical, efficient, and reproducible method that facilitates storing and transferring samples.

Introduction

While measures of oxidative stress and reactive oxygen species are important to the study of diverse diseases across all organ systems, the detection of reactive oxygen species (ROS) is challenging due to a short half-life and high reactivity. An electron paramagnetic resonance (EPR) technique is the most unambiguous method for detecting free radicals. Spin probes have advantages over the more commonly used fluorescent probes. Though fluorescent probes are relatively inexpensive and easy to use and provide rapid, sensitive detection of ROS, they do have serious limitations due to artifactual signals, an inability to calculate ROS concentrations, and a general lack of ....

Protocol

All animal studies were approved by the University of Colorado Denver Institutional Animal Care and Use Committee.

1. Preparation of Reagents

1. Diethylenetriaminepentaacetic acid (DTPA) stock (150 mM)
   1. Add 2.95 g of DTPA (393.35 g/mol) to 10 mL of deionized water.
   2. To dissolve DTPA, add 1 M NaOH dropwise and bring to a pH of 7.0.
   3. Bring the volume to 50 mL with water for a final DTPA concentration of 150 mM, and store at 4 °C.
   .......

Discussion

The assessment of free radical production in biological settings is important in understanding redox regulated signaling in health and disease, but the measure of these species is highly challenging due to the short half-life of free radical species and technical limitations with commonly used methods. EPR is a valuable and powerful tool in redox biology, as it is the only unambiguous method for detecting free radicals. In this project, we demonstrate practical EPR methods for designing experiments and preparing samples .......

Materials

Name	Company	Catalog Number	Comments
DMEM	LifeTech	10566-016	cell culture media
Diethylenetriaminepentaacetic acid (DTPA)	Sigma Aldrich	D6518-5G
sodium chloride (NaCl)	Fisher Scientific	BP358-212	used to prepare 50 mM phosphate saline buffer  according to Sigma aldrish
potassium phosphate dibasic (HK2PO4 )	Fisher Scientific	BP363-500	used to prepare 50 mM phosphate saline buffer  according to Sigma aldrish
potassium phosphate monobasic (KH2PO4 )	Sigma Aldrich	P-5379	used to prepare 50 mM phosphate saline buffer  according to Sigma aldrish
Krebs-Henseleit buffer (KHB)	(Alfa Aesar, Hill)	J67820
Bovine erythrocyte superoxide dismutase (SOD)	Sigma Aldrich	S7571-30KU
Phorbol 12-myristate 13-acetate (PMA)	Sigma Aldrich	P1585-1MG	Dissolve in DMSO
Antimycin A (AA)	Sigma Aldrich	A8674-25MG	Dissolve in Ethanol and store in glass vials(MW used is the averaged molecular weights for four lots)
1-Hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine . HCl (CMH)	Enzo Life Sciences	ALX-430-117-M050
1-Hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine . HCl (CPH)	Enzo Life Sciences	ALX-430-078-M250
1-Hydroxy-4-[2-triphenylphosphonio)-acetamido]-2,2,6,6-tetramethylpiperidine, 1-Hydroxy-2,2,6,6-tetramethyl-4-[2-(triphenylphosphonio)acetamido]piperidinium dichloride ( mito-TEMPO-H)	Enzo Life Sciences	ALX-430-171-M005
1-Hydroxy-2,2,6,6-tetramethylpiperidin-4-yl-trimethylammonium chloride . HCl (CAT1H)	Enzo Life Sciences	ALX-430-131-M250
Heparin	Sagent Pharmaceuticals	NDC 25021-400-10
Diphenyliodonium chloride	Sigma Aldrich	43088
Deferoxamin mesylate salt	Sigma Aldrich	D9533-1G
Critoseal	Leica	39215003
BRAND disposable BLAUBRAND micropipettes, intraMark	Sigma Aldrich	708733	Capillaries
PTFE FRACTIONAL FLUOROPOLYMER TUBING 3/16” OD x 1/8” ID	NORELL	1598774A	Teflon tubing
SILICONE RUBBER STOPPERS FOR NMR SAMPLE TUBES  FOR THIN WALL TUBES HAVING AN OD OF 4mm-5mm (3.2mm TO 4.2mm ID) TS-4-5-SR	NORELL	94987
EMXnano Bench-Top EPR spectrometer	Bruker BioSpin GmbH	E7004002
EMX NANO TISSUE CELL	Bruker BioSpin GmbH	E7004542