α mem

MTFs from five-day postpartum neonates were cultured, as described in other previous studies [23 (link),25 (link)]. First, neonatal testes were dissected and encapsulated in α-minimum essential medium (α-MEM: Welgene, Daegu, Korea). Encapsulated testes were carefully dissected by forceps into six to eight pieces of approximately 2 mm in diameter, and four to five tissue fragments were transferred onto 1.2% (w/v) agarose gel (Sigma–Aldrich, St. Louis, MO, USA) containing 1 mL of α-MEM (Welgene) and 10% (v/v) Knock-out Serum Replacement (Thermo Fisher Scientific, Walthan, MA, USA). Three to four agarose gels were placed in each well of a six-well plate (Corning Inc., Corning, NY, USA). MTFs were incubated under conditions of 34 °C and 5% CO₂, and the medium was changed twice a week for 30 days. NP (Sigma–Aldrich, St. Louis, MO, USA) was dissolved in DMSO and then diluted in the culture medium to the final concentrations of 1, 10, and 50 μM. The concentration of NP was chosen based on other studies [57 (link),58 (link)]. The 30-day MTF cultures were analyzed to confirm whether more than 60% of the seminiferous tubules contained differentiated germ cells for toxicity testing in accordance with other studies [59 (link)].

Bone marrow cells were isolated from tibiae and femurs of 5-week-old Pink1 WT and Pink1 KO mice and cultured overnight in α-MEM (Welgene, Daegu, Korea) containing 1 % penicillin and streptomycin and 10 % fetal bovine serum (hereafter referred to as α-MEM complete medium). BMMs were obtained by culturing non-adherent cells from the bone marrow cell culture with 30 ng/ml of macrophage colony stimulating factor (M-CSF) for 3 days. To obtain pre-osteoclasts (pOCs) and mature osteoclasts, BMMs were cultured, respectively, for 2 and 4 days in α-MEM complete medium with 100 ng/ml of receptor activator of nuclear factor-κB ligand (RANKL) and 30 ng/ml of M-CSF. In cell treatment experiments, BMMs seeded on 48-well plates were cultured with spermidine (5 μM) or NAC (2.5 mM) in the presence of RANKL and M-CSF for 3 days.

The antibody Ab4 was selected from a human naive Fab library, and Ab4M was generated by increasing the affinity of Ab4 45-fold via mutation of three residues in the complementarity-determining regions (CDRs). Next, the human anti-L1CAM mAb Ab417 was generated by increasing the affinity of Ab4M via yeast display of scFv containing randomly mutated light chain CDR3. Ab417 was found to be cross-reactive with mouse L1CAM^{35 (link)}.
The anti-L1CAM antibody Ab417^{35 (link)} was purified from the culture supernatants of transfected CHO cells. Cells expressing Ab417 were cultured in MEM-α (WELGENE) supplemented with 5% (v/v) dialysed FBS (Thermo Fisher Scientific) under 5% CO₂, and the medium was changed to serum-free medium (SFM4CHO, GE Life Sciences) for antibody production. The culture supernatant was centrifuged and filtered using a Sartolab bottle-top filter (0.22 µm, PES; Sartorius) and then subjected to affinity chromatography on a protein A-agarose column (GenScript) for purification. The protein A-bound antibodies were eluted using 0.1 M sodium citrate containing 150 mM sodium chloride (pH 3.6). The eluted antibodies were stored after a buffer change to 25 mM sodium citrate containing 150 mM sodium chloride and 0.007% Tween 20 (pH 6.4). The protein concentrations were determined using a NanoDrop 2000 UV-Vis Spectrophotometer (Thermo Fisher Scientific).