I am Frederick Dina, And I'm an associate professor of pharmacology here at the Ohio State University. In this demonstration, we will show the detection of nitric oxide and superoxide from cells using EPR. Spin Trapping.
Reactive oxygen and nitrogen species have been implicated in the development of various diseases, and therefore their detection in biological systems is of paramount importance. Only few reliable methods for the detection of reactive species have been employed in biological systems, but they are limited by specificity and sensitivity. Spin dropping, however, is commonly used for the identification of eros and involves the addition reaction of radical to a nitro spin trap or metal complexes forming a persistent spin add, which can be detected by electron paramagnetic resonance spectroscopy or EPR, the various radical add ducts exhibit the distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production.
Nitric oxide Syntase catalyzes the conversion of L-arginine to L citruline using N-A-D-P-H to produce nitric oxide. Under oxidative stress conditions, ENA can switch from producing nitric oxide to superoxide in a process called uncoupling, which is believe to be caused by oxidation of heme or the cough factor that hydro terin nitric oxide regulation in biological system is important as it plays critical role in cell signaling resulting in beneficial physiological functions such as vaso dilation, but its overproduction is often associated with inflammatory conditions. Therefore, nitric oxide detection in biological system is important.
Another important class of signaling molecules in biological systems are reactive oxygen species like nitric oxide, reactive oxygen species in low concentrations play an important role in regulating cell function, signaling and immune response, but in unregulated concentrations can lead to cellular oxidative damage leading to oxidative stress. S peroxide radical anion is one of the most important reactive oxygen species and it's the major precursor of some of the most highly oxidizing species known to exist in biological systems such as S peroxide nitrite and hydroxy rod. The detection and or sequestration of S peroxide in biological system is therefore important since its generation signals the first sign of oxidated burst Prior to the start.
This protocol trypsin eyes cultured bovine aortic endothelial cells as described in the written procedure. Transfer the cells onto a labeled six. Well plate and incubate overnight at 37 degrees Celsius and 5%CO2 following incubation.
Remove the six well plate from the incubator and aspirate the medium from the first. Well wash the cells twice with one milliliter delcos phosphate buffer saline or DPBS. Prepare a 1.9 millimolar iron sulfate hep dehydrate solution by adding 0.8 milligrams iron sulfate to one milliliter DPBS with calcium chloride and magnesium chloride.
And prepare MGD solution by adding 2.7 milligrams ammonium MGD to 500 microliters DPBS with calcium chloride and Magnesium chloride. Add 210 microliters of freshly prepared 1.9 millimolar iron sulfate hep dehydrate, and 210 microliters of freshly prepared MGD using a molarity ratio of one to seven and mixer Resulting suspension. Add 4.6 Microliters of calcium ion four and mix the solution again by pipetting up and down.
Then incubate the sample at 37 degrees Celsius for 36 minutes. Collect the supernat that settles In an einor tube. Enter EPR acquisition Parameters center field of 34 27 goss modulation, amplitude of six goss and frequency of 9.8 gigahertz.
Since parameters will vary from one instrument and experimental conditions to another, please see text for other parameters used in this equipment. Transfer the super natin to an EPR flat cell seal with paraffin and place in the EPR cavity. Record the spectra for approximately 21 minutes at 121 scans and 10.4 seconds per scan after the experiment has run.
Use brucker WIN EPR processing to correct the baseline and integrate the 2D spectra, which reduces the background noise. It should be noted that the scan time and number of scans can vary from one experiment to another. Also, methods to correct the baseline can also vary from one software To another.
To Begin Enos uncoupling, Remove the medium from the second well of the six. Well plate and wash the cells twice with one Milliliter DPBS. Then Add 100 microliters of the prepared 0.5 millimolar Syn one solution, and bring the volume up to two milliliters with DPBS containing 10%FBS incubate The cells for two hours at 37 degrees Celsius and 5%CO2 following incubation.
Wash the cells with DPBS twice. Then add 210 microliters of freshly prepared, 2.8 millimolar iron sulfate heta hydrate, and 210 microliters of 19.6 millimolar MGD prepared freshly according to the previous procedure. Next, add 4.6 microliters of 1.9 millimolar calcium ion four, and mix the solution again.
As before, incubate the cells at 37 degrees Celsius for 36 Minutes. After 36 minutes, collect the super natin that settles in an einor tube and transfer to an EPR flat cell. Record the EPR spectra using the same acquisition parameters as before following acquisition.
Process the data in the same Manner prior to the start of this Protocol. Prepare a stock solution of one molar DMPO and PBS containing 0.1 millimolar DTPA. Then make a one milligram per milliliter solution of four by 12.
Myra State 13 acetate or PMA in DMSO make aliquots of one milliliter of the PMA stock solution by diluting it to 10 micrograms per milliliter in PBS in an einor tube containing one milligram per milliliter D glucose and one milligram per milliliter. Albumin makes five to 6 million PMN cells per milliliter by pipetting up and down in a 1.5 milliliter einor tube. Prepare a mixture with a final volume of 60 microliters containing approximately 1.6 times 10 to the six cells of PMN 10 millimolar DMPO and 0.2 micrograms per milliliter of PMA.
In this experiment, we will be using a Bruker ESP 300 X band spectrometer. Transfer the Solution to an EPR flat cell, then place the flat cell in the EPR cavity. Enter EPR acquisition parameters center field of 3, 486 goss modulation amplitude of 0.5 goss, and frequency of 9.8 gigahertz.
Since parameters will vary from one instrument to another and experimental conditions to another, please see text for the other parameters used in this experiment Acquire spectra. In this demonstration, we show the detection of nitric oxide and SU peroxide radicals using two different spin traps. The iron MGD and DNPO.
We show that stimulation of endothelial cells with calcium ion four produces nitric oxide and the treatment with seen one. A perine nitrite donor can decrease nitric oxide production, a stimulation of neutrophils, however, with PMA in the presence of a nitro spin trap gives a hydroxyl radical ADDA instead of the S oxide due to the instability of the initially formed superoxide adda. While the mechanisms of spin trapping by iron mg d nitros are fundamentally the same, the nitric oxide addition to iron MGD involves pass rate of reaction and that the iron MGD nitric oxide add form is very stable.
That in fact one can freeze the solution at minus 80 degrees centigrade for later acquisition of the EPR spectrum without significant decrease in its EPR signal intensity. On the other hand, the detection of oxide using the nitro spin trap is slow. That's requiring higher concentrations of the spin trap compared to the counterpart iron MGD four nitric oxide detection.
Several approaches have been employed to successfully detect the superoxide adduct form, which we did not demonstrate anymore in this video. The use of flat cell, for example, can give better su peroxide add signal intensity due to higher cell density. I encourage you to refer to the text on this matter.
Also, instead of using DMPO, one can employ a certified spin trap such as BMPO and VMP or the phosphorylated Nitro DAP MPO, which can yield more persistent SU peroxide addax. The use of cyclodextrin has also been employed to stabilize SU peroxide add adapt when employing spin trapping for US detection. Immediate acquisition of the EP spectrum after SU superoxide generation is highly recommended.
Therefore, the detection of nitric oxide using iron MGD is much more efficient and the major challenge now that confronts a PR spin trapping of superoxide is to attain those characteristics observed for the formation of iron MGD nitric oxide addoch. That is fast, superoxide reactivity and stable add efforts in the development of spin traps with fast reactivity to superoxide and formation of stable spin. ADD is now being carried out in our laboratory and by other Groups as well.
