Drabkin s reagent

The hemolysis assessment was performed as previously described [45 (link)]. Briefly, frozen whole blood and supernatant samples were fully thawed. An aliquot of each supernatant sample was transferred to a fresh tube and centrifuged at 3000 × g for 3 min. Packed, unwashed blood samples from day 1 of storage were vortexed for 30 s and diluted 1:10 with deionized (DI) water. Finally, 10 μl of each sample was transferred to a 96‐well flat bottom plate (BioVision Inc) together with 100 μl of Drabkin's reagent (MilliporeSigma) containing Brij‐35 solution (Thermofisher). The plate was then incubated at room temperature for 15 min on a plate shaker, and absorbance was read on a microplate reader (manufacturer) at 540 nm. Hemolysis in the supernatant at each timepoint was calculated relative to whole blood at day 1, using the following formula:
$$\%\mathrm{Hemolysis}=\frac{\mathrm{O}{\mathrm{D}}_{\mathrm{supernatant}}\times \left(1-\mathrm{Hct}\right)}{\mathrm{O}{\mathrm{D}}_{\mathrm{whole}\mathrm{blood}}}\times 100\%$$ where Hct is the hematocrit, and ODsupernatant and ODwholeblood are the measured optical density from the supernatant and whole blood, respectively.

The concentration of hemoglobin in the residual hematoma was detected using Drabkin's reagent (Merck Millipore, Germany) according to the manufacturer's instructions. Briefly, the residual hematoma samples were collected and homogenized. After centrifuging the homogenate, the supernatant was mixed with the reagent and incubated for 20 minutes. The absorbance was detected with a microplate reader, and the concentration of hemoglobin was calculated from a standard curve.

Mice were sacrificed at 1 dpi and perfused with heparinised saline to remove intravascular blood. A freshly dissected spinal cord (epicentre ± 2.5 mm) was homogenised in distilled water (250 μL) and processed to measure tissue bleeding, as previously described^{38 (link)}. Briefly, 20 μL of supernatant containing haemoglobin was incubated with 80 μL of Drabkin’s reagent (Merck KGaA), and the haemoglobin concentration was assessed by measuring the optical density of the solution at a 550 nm wavelength.

At T0 and at T2, blood samples were collected from central venous or arterial access and centrifuged to obtain plasma and serum samples that were stored at – 80 ℃ for subsequent analysis. 8-Epi-prostaglandin F2-alpha was analyzed as marker of oxidative damage; syndecan-1 and glypican-3 as markers of damage of glycocalyx; endothelin-1 as marker of endothelial damage.
Cell-free Hb and nitric oxide were quantified in plasma through colorimetric assay (Drabkin’s reagent—Sigma-Aldrich, Saint Louis, Missouri, USA; Nitric Oxide Assay kit—Elabscience, Houston, Texas, USA).
Concentrations of syndecan-1 (Human SDC1 immunosorbent assay [ELISA]), glypican-3 (Human GPC3 ELISA kit), 8-epi-prostaglandin F2-alpha (8-epi-PGF2α ELISA kit) were measured in serum and endothelin-1 (Human ET-1 ELISA Kit, Elabscience, Houston, Texas, USA) was determined in plasma, in both cases by using the corresponding enzyme-linked immunoadsorbent essay (ELISA) kits (Elabscience, Houston, Texas, USA) in accordance with the instructions of the manufacturer.

As described in previous studies, hemoglobin levels were quantified to determine the residual hematoma volume 10 (link). Following euthanasia, rats were transcardially perfused with ice cold PBS to remove circulating blood. The collected meninges with hematomas were ground, ultrasonicated, and centrifuged (13000 RPM, 30 min) in 1 mL of PBS. A 0.4 mL aliquot of the resulting supernatant was mixed with 0.6 mL of Drabkin's reagent (Sigma-Aldrich, cat no. D5941). After a 15-minute reaction at RT, 200 µL of the mixture was added to a 96-well plate. The absorbance of each well was measured at a wavelength of 540 nm using a microplate reader. Autologous blood was also processed similarly to establish a standard curve with eight concentrations ranging from 0 to 400 µL. The residual SDH volume was calculated based on the absorbance values using Excel.

Hematology was measured both before and after hypoxia acclimation. Blood samples were taken from the facial vein 3 days after V̇O₂max measurements. We measured Hb content using Drabkin’s reagent (according to the instructions from the manufacturer, Sigma-Aldrich) and hematocrit by spinning the blood in capillary tubes at 12,700g for 5 min. The O₂ affinity of intact erythrocytes was measured using 10 μl blood in 5 ml buffer containing 0.1 M Hepes, 0.05 M EDTA, 0.1 M NaCl, 0.1% bovine serum albumin, and 0.2% antifoaming agent at pH 7.4. Oxygen dissociation curves were generated at 37 °C using a Hemox Analyzer (TCS Scientific), and red blood cell P₅₀ and Hill coefficient (n) were calculated using the Hemox Analytic Software.