Orion perphect ross combination ph micro electrode

The pH of the sole aqueous otic product (Baytril Otic) was measured using a pH meter (Thermo Scientific Orion PerpHecT ROSS™ Combination pH Micro Electrode). The reading was performed in triplicates. The results were reported as mean and standard deviation.

1X PBS was pH adjusted using 0.1 M hydrochloric acid or 0.1 M sodium hydroxide. A pH probe (Orion PerpHecT ROSS Combination pH microelectrode, Thermo Scientific) was calibrated at pH 2, 4, 7, and 11 used to monitor the solutions.

Fresh whole blood was diluted 1:15 with each individual’s own plasma, and O₂ equilibrium curves were measured immediately after sampling. To obtain stripped hemolysate, 100 μL of centrifuged red blood cells were added to a 5× volume of 0.01 M Hepes/0.5 mM EDTA buffer (pH 7.4), followed by a 30-min incubation on ice to lyse the red blood cells. NaCl was added to a final concentration of 0.2 M, and samples were centrifuged at 20,000 × g for 10 min to remove cell debris. Hemolysate supernatants and purified recombinant Hb were similarly desalted by passing through a PD-10 desalting column (GE Healthcare) equilibrated with 25 mL of 0.01 M Hepes/0.5 mM EDTA (pH 7.4). Eluates were concentrated using Amicon Ultra-4 Centrifugal Filter Units (EMD Millipore). From these concentrated samples, Hb solutions (0.1 mM Hb in 0.1 M Hepes/0.05 M EDTA buffer) were prepared in the absence (stripped) and the presence of 0.1 M KCl and 0.2 mM inositol hexaphosphate (+KCl +IHP). Stripped and +KCl +IHP treatments were prepared at three different pH values (for a total of six treatments per Hb sample). Working solutions were adjusted with NaOH to as close to pH 7.2, 7.4, or 7.6 as possible, and then pH was precisely measured with an Orion Star A211 pH Meter and Orion PerpHecT ROSS Combination pH Micro Electrode (Thermo Fisher Scientific).

Vertical oxygen and hydrogen sulfide concentration profiles along the top layer of the gradient tubes were measured using Unisense (Aarhus, Denmark) OX-100 and H2S-100 microsensors (respectively). The oxygen microsensor was calibrated using freshly prepared de-oxygenated water (0% oxygen saturation) as well as a well-aerated water (100% oxygen saturation). The hydrogen sulfide solution was calibrated using freshly prepared de-oxygenated solutions of Na₂S in an acetate buffer (pH <4), in the 0–500 μM H₂S range. The microsensors as well as a calibrated pH micro-electrode (Thermo Scientific Orion PerpHecTROSS Combination pH Micro Electrode) were mounted on a motor to measure vertical H₂S, O₂ and pH profiles in the top layer of the gradient tubes with a 2 mm step. We checked that profile measurements did not significantly perturb the chemical gradients in the tubes by measuring the same tubes after a few minutes interval and obtaining identical profiles. H₂S and pH measurements were used to calculate total sulfide concentrations in the tubes using the acid dissociation constant for H₂S/HS⁻ from ref. 50 .

Osmolality of testicular fluid (n = 5 males) was determined in triplicate for each sample using a Vapor Pressure Osmometer (Model 5600 Wescor, Inc, Logan, Utah, USA). The pH of testicular fluid (n = 5 males) was measured using a benchtop pH meter (Orion Star™ A111 Benchtop pH Meter, Thermo Fischer Scientific, Madison, WI, USA) and an electrode (Orion™ PerpHecT™ ROSS™ Combination pH Micro Electrode, Thermo Fischer Scientific, Madison, WI, USA). Osmolality and pH were also measured for all activation media.