DMPO

This plan only provides a guide, please modify it to meet your specific needs.

1. Solution preparation

(1)Working solution: Dissolve DMPO in experimental buffer to a final concentration of 1 M. Use 0.22μm filter film filter.

Note: Please adjust the optimal working concentration according to the actual situation or refer to the literature to set the gradient concentration by yourself. The working solution must be prepared and used immediately.

2. In vivo detection of oxygen free radicals using DMPO ^{[1, 2]} (from the literature, for reference only)

(1)Induce intraperitoneal anesthesia with sodium pentobarbital (5 mg). Remove the heart, connect the ascending aorta cannula, and perfuse with Krebs-Henseleit buffer at a constant flow rate (2 mL/min).

(2)After ischemia, dissolve DMPO in a buffer containing 100μM diethylenetriaminepentaacetic acid (DTPA) to a final concentration of 1 M. Immediately during reperfusion, inject the trap at a rate of 100μL/min through the side arm close to the heart.

(3)During reperfusion, collect the effluent regularly until reperfusion for 5 minutes at the specified time interval. After collecting the samples, freeze each tube in liquid nitrogen immediately.

(4)Use a spectrometer to detect and record the signal of oxygen free radicals captured by DMPO.

3. In vitro detection of hydroxyl free radicals using DMPO ^[3] (from the literature, for reference only)

(1) Add different concentrations of Fe3O4 solution to 1 mL of sample. Add 10 μL of DMPO working solution to each sample as a free radical scavenger. Incubate the sample at 37°C for 6 hours to ensure that the reaction is fully carried out. Add 10 μL of DMPO working solution to 1 mL PBS as a control group of the reaction solution.

(2) The reaction mixture was filtered using a 0.22 µm filter to remove solid residues and large particles.

(3) Use a spectrometer to detect and record the signal of hydroxyl free radicals captured by DMPO.

Note: This protocol provides guidance for the detection of oxygen-centered free radicals using DMPO in vivo and in vitro. It can be adjusted according to other literature and specific experimental requirements.

References:

[1]West M B, Rokosh G, Obal D, et al. Cardiac myocyte–specific expression of inducible nitric oxide synthase protects against ischemia/reperfusion injury by preventing mitochondrial permeability transition[J]. Circulation, 2008, 118(19): 1970-1978.

[2]Wang P, Chen H, Qin H, et al. Overexpression of human copper, zinc-superoxide dismutase (SOD1) prevents postischemic injury[J]. Proceedings of the National Academy of Sciences, 1998, 95(8): 4556-4560.

[3]Qin X, Wu C, Niu D, et al. Peroxisome inspired hybrid enzyme nanogels for chemodynamic and photodynamic therapy[J]. Nature Communications, 2021, 12(1): 5243.