F12 culture medium

Human umbilical cords were obtained from healthy women who had childbirth in our hospital. All samples obtained were provided by these women who agreed and signed consent forms. C57BL mice (6–8 weeks) were ordered from Beijing Vital River Laboratory Animal Technology Co. Collection of samples and handling animals were all approved by the Ethics Committee of Neurology Department of General Hospital of Jinan Military Region. All experiments were reviewed by the Ethics Committee of Neurology Department of General Hospital of Jinan Military Region. Adenovirus expressing green fluorescent protein gene (Ad-GFP) was provided by Beckman Medical Instruments, USA. Recombinant adenovirus carrying FGF-20 gene (Ad-FGF-20) was constructed in our lab.
Consumables such as F12 culture medium, pipettes, and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were all ordered from Sigma (USA). Fetal calf serum was ordered from HyClone Company (USA). All ELISA reagent kits were ordered from IBL (Germany). All antibodies were ordered from Abcam (England). Chromogenic substrate kit was ordered from Shanghai Ruichi Biotechnology Co., Ltd. (Shanghai, China).

Tunica albuginea was cut and peeled back to expose tubules. Ten fragments of SeT were placed in culture plate (Nunclon D 12 well multidishes; Nunc, Roskilde, Denmark) wells containing 2 ml of pre-warmed DMEM: F-12 culture medium (Sigma-Aldrich) supplemented with 20 mg/L gentamicin sulfate, 0.1 mM 3-isobutyl-1-methylxanthine, and 1 µg/L BSA. SeT fragments of 1 cm were used to guarantee the morphometric homogeneity of testicular cell types. The SeT from Wt and Tg-RGN rats were incubated for 48 hours at 33 °C in an atmosphere of 5% CO₂. At the end of the experiment, culture medium and SeT were recovered, snap–frozen in liquid nitrogen and stored at −80 °C.

Umbilical cord tissues were obtained from healthy pregnant women admitted to our hospital for delivery. These women signed a written informed consent. C57BL mice (6-8 weeks) were ordered from Beijing Vital River Laboratory Animal Technology Co. (China). Consumables such as F12 culture medium and pipettes were ordered from Sigma (St. Louis, USA). CUR, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and MPP+ were ordered from Sigma (St. Louis, USA). Fetal calf serum was from HyClone (Waltham, USA). All ELISA kits were from IBL (Germany). All antibodies were purchased from Abcam (UK), BD Biosciences (San Jose, CA), BD (Franklin Lakes, NJ), and BioLegend (Cambridge, UK). Chromogenic kits were ordered from Shanghai to Ruicheng Biotechnology Co. Ltd (Shanghai, China). The SH-SY5Y cell line was obtained from the Shanghai Institute of Cell Biology of the Chinese Academy of Sciences.