Model 22 syringe pump

MS analysis of the irradiated rose bengal/2-thioHis and rose bengal/EGT samples were completed to determine what oxidized species were present. MS analysis of oxidized 2-thioHis and EGT samples with rose bengal in D₂O was completed within 30–45 min of irradiating the samples to avoid significant decomposition of any oxidation products. For these analyses, an Applied Biosystems QTrap 4000 hybrid triple-quadrupole/linear ion trap LCMS (SciEx, Framingham, MA, USA) was used. Positive electrospray ionization (ESI) was used as the ionization source. Samples were diluted with deionized water and directly infused via a Harvard Apparatus model 22 syringe pump (Harvard Apparatus, Holliston, MA, USA) at 5 µL/min into an isocratic (50% water and 50% acetonitrile with 0.1% formic acid) mobile phase flow from a Shimadzu Prominence high-performance liquid chromatography (HPLC) system (Shimadzu Scientific Instruments, Columbia, MD, USA). Mobile phase flow was maintained at 100 µL/min. Source temperature was maintained at 400 °C. Nitrogen was used for the sheath gas, auxiliary gas, and curtain gas. Sheath gas (GS1) flow was set at 40, auxiliary gas flow (GS2) at 50, curtain gas flow (CUR) at 30, and the declustering potential (DP) was set to 50. The mass spectrometer was operated in single quadrupole mode, scanning from m/z 100 to 1000.

We performed microdialysis as described previously (Friston et al., 2019 (link)). Briefly, sterile linear microdialysis probes (400 μm 3 MDa cut-off microdialysis catheters; Dermal Dialysis) were inserted into subcutaneous tissue of the dorsal aspects of both hind limbs. The probes were placed just below the dermis. The active uptake length of the probes (i.e. the length of the probes through which molecules from the interstitium could enter the perfusate) was 10 mm. Probe insertion was performed 50 min before the burn injury to allow for 20 min of fluidics equilibration and a further 30 min ‘flush’ period (Fig. S2). The latter enabled comparison of pre-burn cytokine levels within each animal and further allowed stabilisation of any inflammatory response resulting from probe insertion. The probes were connected to a Model ‘22’ syringe pump (Harvard Apparatus) using plastic tubes and Ringer's solution (Baxter) was perfused through the tubes and connected probes at a rate of 2 μl/min. Microdialysate was collected for 0.5 h pre-burn and 3 h post-burn in half hour fractions. Collected microdialysates were stored at −80°C until analysis.