Glutathione assay kit

In the present experimental study, 40 adult NMRI mice (8 wk, 25–30 gr) were purchased from the experimental animal center, Royan Institute, Tehran, Iran, and were kept in a clean cage with control condition according to the National Institutes of Health guideline (12 hr light/dark cycle, room temperature: 22 $\pm$ 3 C). All animals had free access to water and food.
Dulbecco's Modified Eagle Medium F12 (DMEM), fetal bovine serum, and lysis buffer were purchased from ABCAM (Cambridge, UK). DCFDA, trypsin, collagenase, and Hams F-10 were obtained from sigma Aldrich (St. Louis, MO, USA). Polymerase chain reaction kit was purchased from Takara (TAKARA, Bio, Japan). SCD kit was obtained from Indas labratorios (INDAS Lab, Madrid, Spain). DNaseI was purchased from Roche (Roche, Basel, Switzerland). Glutathione assay kit was obtained from Zellbio (Zellbio, Lonsee, Germany). Other materials were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

The ROS content of testicular tissue was measured using dichlorofluorescin diacetate (DCFDA: CAS Number 2044–85-1, Sigma-Aldrich, USA) and spectrofluorimetric method. In this test, 50 mg of tissue was mixed with 100 μL of 20 μM DCFDA. The samples were then incubated for 45 min at 37 C in the dark. After incubation, the testicular tissue was cut into small pieces with a sonicator. Next, the lysed samples were centrifuged at 1500 rpm for 5 min at 4 C. Then, the supernatant of the samples was checked using a spectrofluorimeter with a wavelength of 488 nm. To analyze the data with logarithmic concentrations, 5 concentrations were calculated and a standard graph was drawn. Other samples were evaluated using a standard chart. The ROS level was determined as mM H ${\hspace{0.17em}}_2$ O ${\hspace{0.17em}}_2$ based on the standard curve. According to the manufacturer's instructions, Glutathione peroxidases was determined using a glutathione assay kit (Zellbio, Lonsee, Germany). 1 molar of GSH is decomposed by 1 Glutathione peroxidases activity unit in 1 min. At 495 nm excitation and 530 nm emission wavelengths, 2 markers named O-phthalaldehyde and N-Ethylmaleimide were applied in the test to determine the ratio (16).