Phosphate buffer 7.4 ph

Glutathione reductase (GR) activity was assessed in peritoneal, spleen and thymus leukocytes following a method previously described [84 (link)] with some modifications [85 (link)]. Aliquots of leukocytes adjusted to 1 × 10⁶ cells/mL in Hank´s solution were centrifuged at 1000 g for 10 min at 4 °C and the supernatants were removed. Thereafter, leukocyte pellets were resuspended in 200 μL of 50 mM phosphate buffer 7.4 pH (Sigma-Aldrich) plus 6.3 mM ethylene-diaminetetracetic acid (EDTA, Sigma-Aldrich), previously degassed. Then, cells were sonicated and centrifuged at 3200 g for 20 min at 4 °C, and aliquots of the supernatant were used to evaluate GR. This method is based on the oxidation of β-NADPH (β-nicotinamide adenine dinucleotide 2-phosphate reduced) (6 mM, Sigma-Aldrich) due to the reduction of GSSG (80 mM, Sigma-Aldrich) by GR. The reaction was followed by spectrophotometry at 340 nm for 240 s. The results were expressed as milliunits (mU) of enzymatic activity per 10⁶ leukocytes.

Catalase (CAT) activity was determined in peritoneal, spleen and thymus leukocytes following a method previously described [83 (link)], with slight modifications introduced by our laboratory [66 (link)]. Aliquots of the three leukocyte samples adjusted to 1 × 10⁶ cells/mL in Hank´s solution were centrifuged at 1000× g for 10 min at 4 °C and the supernatants were removed. Thereafter, leukocyte pellets were re-suspended in 100 μL of 50 mM phosphate buffer 7.4 pH (Sigma-Aldrich), previously degassed. Then, cells were sonicated and centrifuged at 3200× g for 20 min at 4 °C. Aliquots of the supernatant were used to evaluate CAT. The enzymatic assay was followed using a spectrophotometer for 80 s at 240 nm through the decomposition of 14 mM hydrogen peroxide (H₂O₂; PANREAC, Barcelona, Spain), in phosphate buffer, into H₂O and O₂. The results were expressed as International Units (IU) of enzymatic activity per 10⁶ leukocytes.