Emxmicro

HL-60 cells (5 × 10⁶/500 μl of HBSS) were incubated for 20 min with 5 μl of either 5- or 16 DSA (0.01 M). The cells were then centrifuged and the pellets were resuspended in 500 μl HBSS supplemented with either 10⁻⁴ M NDS27, NDS28, 5 × 10⁻⁴ M HPβCD or γ-CD and incubated for 10 min at RT. Subsequently, cells were centrifuged and the pellet (resuspended in 500 μl of HBSS) and supernatant were transferred into separate micro-caps tubes (sealed with rubber and transferred into ESR tube), placed into the cavity of the ESR spectrometer. The ESR characteristic spectra of the 5-DSA and 16-DSA signals were recorded. The measurements were implemented at 300 K with a continuous wave spectrum EMX-micro of Bruker (Brüker, Rheinstetten, Germany), operating at fixed X-band frequency of 9.5 GHz and at a microwave power of 10.88 mW. The instrumental settings were as follows: 100 kHz modulation frequency, 2 G modulation amplitude, 3389.698 G magnetic field center, and 3.56 × 10⁴ receiver gain. The sweep width was 89.652 G and the total number of scans was five.

This experimental procedure was based on methods previously reported with slight modification.^[61 ,
⁷² (link)
^] Briefly, 0.5 mM GSH solution was mixed with different concentrations of Lac‐FcMOF. After stirring for various durations (1 h, 4 h, or 10 min) under different conditions, the GSH content was quantified using a reduced GSH content assay kit according to the manufacturer's guidelines. Fenton catalytic activity was characterized through MB degradation experiments and ∙OH capture using a UV–vis spectrophotometer (Shimadzu 1750, Shimadzu, Japan) and an ESR spectrometer (EMXmicro, Bruker, Germany), respectively. For MB degradation experiment, the concentration of MB was 10 µg mL⁻¹, Lac‐FcMOF was 100 µg mL⁻¹, H₂O₂ was 10 mM, and GSH was 10 mM. For ∙OH capture experiment, 10 µL of DMPO was used as a radical trap, which was added into the suspension containing H₂O₂ (10 mM) or H₂O₂ (10 mM) + GSH (10 mM) + Lac‐FcMOF (100 µg mL⁻¹).

EPR spectroscopy was conducted on a spectrometer (EMXmicro, Bruker, Germany) to identify the radical species during photoreaction. A homogeneous solution was prepared by dissolving KCD (175 µM), TA (3.5 mM), and DMPO (the radical capturing agent, 3.5 mM) in toluene upon bulk ultrasonication (298 K, 30 min). The concentrations of KCD and TA were maintained to be identical to that during the nanosecond transient absorption characterization, respectively. Prior to measurement, oxygen was removed by continuous argon gas purge. Then, the solution was loaded in an EPR columnar quartz cell that was subsequently placed into the spectrometer. Radicals were generated upon exposure to light irradiation under a high-pressure mercury lamp and their signals were in situ recorded. The light wavelength was confined to be 420–780 nm. The applied parameters were set as follows: center field, 3514.2 Gauss (G); sweep width, 100 G; microwave power, 2.0 mW; modulation frequency, 100 kHz; modulation amplitude, 1 G; receiver gain, 30 dB; conversion time, 15 ms; time constant, 0.01 ms. The computer program EasySpin^{61 (link)} was employed to fit the recorded signals.

Intracellular and extracellular concentrations of ROS and reactive nitrogen species (RNS) were determined in lung homogenate by a Bruker electron spin resonance (ESR) spectrometer (EMXmicro; Bruker Biospin, Germany), using the spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH [40 (link)]). Lung tissue were homogenized in ESR-Krebs HEPES buffer (99.0 mM NaCl, 4.69 mM KCl, 2.5 mM CaCl₂x 2H₂O, 1.2 mM MgSO₄ x 7H₂O, 25 mM NaHCO₃, 1.03 mM KH₂PO₄, 5.6 mM D(+) Glucose, 20 mM Na-HEPES, 25 μM deferoxamine, 5 μM diethyldithiocarbamate) containg protease inhibitor cocktail (Sigma-Aldrich, USA). Afterwards, samples were incubated for 30 min at 37°C with 0.5mM CMH. Following this procedure samples were collected into 1 ml syringes and flash frozen in liquid nitrogen. The X-Band (9.65 GHz) ESR measurements were performed at room temperature (20 - 22°C). The experimental parameters were as follows: G-factor 2.0063, center field ~3360 G, microwave power 2,000 mW, receiver gain 50 dB, time constant 10,24 ms, modulation amplitude 2,999 G, modulation frequency 100 GHz. The data from ESR microscopy were normalized to protein concentration measured by DC protein assay from Bio-Rad.

All cw EPR experiments were conducted at X-band frequency on a Bruker EMXmicro (Bruker, Billerica, MA, USA) EPR spectrometer with the EMX standard resonator (4119HS).

For the detection of ^•NO in solution, electron paramagnetic resonance (EPR) spectroscopy measurements were performed. An X-band (9.87 GHz) spectrometer (EMXmicro, Bruker) was used with a modulation frequency of 100 kHz, modulation amplitude of 0.1 mT, microwave power of 5.024 mW, receiver gain of 30 dB, and time constant of 0.01 ms. To detect ^•NO, a nitronyl nitroxyl radical (NNR) spin trap (Carboxy-PTIO (2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, Dojindo Laboratoire), was used at 60 μM, which forms an imino nitroxyl radical (INR) with ^•NO. BMPO (5-tert-Butoxycarbonyl-5-methyl-1-pyrroline-N-oxide, also Dojindo) was used as a spin trap for sulfur, hydroxyl, and superoxide anion radicals. For some experiment, 60 μM Carboxy-PTIO were mixed with 2 mM BMPO immediately before use. The spin trap solutions were freshly prepared prior each experiment and untreated samples were measured as respective controls. The maximum handling delay between treatment and measurement was three minutes. The spectra were recorded and evaluated by Bruker’s Xenon software. More details about the measurement procedure and evaluation of the spectra can be found in a previous publication [47 (link)].