Dysregulated redox signaling is implicated in the pathogenesis of diverse diseases. The study of redox biology requires the accurate detection of reactive oxygen species in different cellular and tissue compartments. EPR spectroscopy is the only method that measures free radicals unambiguously.
This protocol demonstrates a practical method for handling and storage of biologic samples for EPR spectroscopy. These protocols illustrate the use of EPR and spin probes in two representative models. Although they can be adapted to evaluate the active species in other biological settings.
The design of the experiment and the handling of the sample are critical for its reproducibility. We recommend considering cell density and ensuring appropriate time-matched controls. Begin by gently washing raw 264.7 cells with one milliliter of Krebs-Henseleit Buffer or KHB, per well.
And treat the cells with 500 microliters of KHB, supplemented with 100 micromil of DTPA per well. To pretreat with superoxide dismutase, add 15 microliters of superoxide dismutase stock, or vehicle, to each well, and place the plate at 37 degrees Celsius for 10 minutes. At the end of the superoxide dismutase pretreatment, add 12.5 microliters of 10 millimolar CMH and 40 microliters of 125 micromolar PMA working solution to the appropriate wells, and incubate at 37 degrees Celsius for 50 minutes.
Immediately place the plates on ice and collect the buffer from each well into individual 1.5 milliliter tubes on ice. Next, add 100 microliters of fresh KHB plus DTPA to each well and gently scrape the cells from the bottom of each well. Then resuspend the cell suspensions with pipetting and transfer the cells into individual 1.5 milliliter tubes on ice.
For superoxide detection at room temperature, first load 50 microliters of one buffer or cell sample into a capillary tube and seal the tube. Immediately place the tube into the electron paramagnetic resonance, or EPR, spectrometer and set the EPR acquisition parameters for room temperature measurements. Always test a blank sample of buffer containing the same concentration of probe treated under the same experimental conditions as a control.
For superoxide detection at 77 degrees Kelvin, load 150 microliters of one sample into a one to two inch piece of straight polytetrafluoroethylene, or PTFE, tubing, with a rubber stopper at one end. Seal the other end of the tube with a second stopper and flash freeze the sample in liquid nitrogen. Then quickly remove the stoppers and place the PTFE tubing in a labeled cryotube for liquid nitrogen storage.
For EPR measurements, fill the finger dewar of the EPR spectrometer with liquid nitrogen and place the PTFE tubing containing the sample in the liquid nitrogen. Place the finger dewar into the EPR spectrometer and set the EPR acquisition parameters for measurements at 77 degrees Kelvin. For ROS detection in frozen lung tissue, place the tissue on dry ice and use tweezers to stabilize the flash-frozen tissue on dry ice.
Cut the sample into five 15 milligram pieces, and weigh the pieces in a tared 1.5 milliliter tube. Add 196 microliters of KHB plus DTPA and four microliters of CMH to the sample, and incubate the lung sample for one hour in a 37 degree Celsius water bath before pelleting the tissue fragments with a brief centrifugation. Then place the sample on ice before transferring 150 microliters of the supernatant into a piece of PTFE tubing for flash freezing in preparation of EPR measurement as just demonstrated.
Evaluation of the total superoxide production in raw 264.7 cells stimulated with PMA as demonstrated, reveals that the nitroxide concentration in both the cell suspension and the buffer of the PMA-treated cells is similar, due to the permeable nature and rapid equilibration of the spin probe. The nitroxide radicals signal increases in raw 264.7 cells stimulated with PMA, compared to control cells, but not in cells pretreated with cell-impermeable superoxide dismutase. The concentration of nitroxide obtained at low temperatures in raw 264.7 cells after stimulation with PMA is also attenuated in the presence of superoxide dismutase consistent with the room temperature data.
The nitroxide concentration is increased in the blood and bronchoalveolar lavage fluid of bleo mice and treated mice, as well as within the supernatants of harvested lung tissue pieces from bleo mice and injured animals, compared to PBS-treated controls. Further, after retro-orbital spin probe injection, bleo mice and treated animals demonstrate a higher spin probe signal compared to controls. These practical protocols will allow researchers to test free radical production in different cellular compartments and biological samples by EPR, including low temperature measurements.
