X band epr instrument

To determine the intracellular oxygen content, rat cardiac fibroblasts were incubated with lithium phthalocyanine (LiPc) nanoparticles for 2 h to allow cellular uptake. The residual LiPc nanoparticles were washed with DPBS for 3 times. After trypsinization, the cells were seeded in collagen-coated EPR tubes (Wilmad-LabGlass, n = 5 for each group) with or without PCNP/O₂ (10 mg/mL). The EPR tubes were placed in a hypoxic incubator (1% O₂, 37 °C) for 4 h for gas balance. After that, the tubes were sealed and incubated for 24 h under 1% O₂. The EPR spectrum was recorded using an X-band EPR instrument (Bruker). The parameters used in this experiment were 0.1 mW for microwave power, 1.0 db for attenuation, and 9.8 GHz for frequency, following our reported method.^{70 (link)} Oxygen content (%) was calculated from the line width of the spectrum using a calibration curve.

To test oxygen preservation capability of the hydrogels, the oxygen partial pressure (pO₂) in the hydrogels at 21% and 1% O₂ conditions were measured using electron paramagnetic resonance (EPR). Oxygen sensitive lithium phthalocyanine (LiPc) was used as EPR probe [61 ]. LiPc particles (<1 μm in diameter, 40 mg/mL in DPBS) were thoroughly mixed with 8% hydrogel solution that was stirred under atmospheric condition (21% O₂) overnight. 200 μL of the mixture was transferred to a 37 °C water bath for gelation. After 30 min, the solid gel was loaded into a gas permeable EPR tube under atmospheric condition (21% O₂). The EPR spectrum was recorded on an X-band EPR instrument (Bruker). The parameters used included 0.1 mW for microwave power, 9.8 GHz for frequency and 1.0 dB for attenuation. After measurement under 21% O₂, the sample was flushed with 1% O₂ to reach equilibrium as monitored by EPR. The EPR spectrum was then recorded. LiPc loaded with DPBS was used as control. pO₂ was calculated based on linewidth of the sample and calibration curve of linewidth vs. oxygen concentration. Four replicates for each hydrogel type were tested.