High glucose dmem f12

Nile tilapia SSCs were obtained through primary culture from adult fish testis12 (link)22 23 (link)24 , and 10⁶ cells per dish were exposed to 1 mL of lentiviral solution (titre = 6.5 × 10⁵ TU/mL). The cells were incubated at 33 °C and 5% CO₂ for 24 h, and the aliquot was submitted to flow cytometry in order to assess the percentage of fluorescent cells before selection. Blasticidin (6 μg/mL) was added to the supplemented DMEM/F12 culture medium (High Glucose DMEM/F12 (THERMO), 10% foetal bovine serum, 0.1 mM non-essential amino acids, 6 mM L-glutamine, 1 mM sodium pyruvate, and 1% penicillin-streptomycin) and the cells were maintained for one month12 (link). After this time, their fluorescence was assessed by flow cytometry. Cell genomic DNA was extracted and submitted to PCR in order to confirm the presence of the DsRed2 encoding sequence on a 1% agarose gel with Syber Safe (LIFE TECHNOLOGIES). Twenty larvae (2–3 days after hatching) were anesthetised and microinjected in the coelomic cavity with approximately 2 μL of cell suspension containing 5 × 10⁴ SSCs/μL, using a micromanipulator (NARISHIGE) and glass needles prepared with GD-1 capillaries (NARISHIGE) as previously described25 (link). The red fluorescence was monitored through fluorescence and confocal microscopy, and the sperm samples from male recipients were analysed by flow cytometry and fluorescence microscopy.

MCF10A, MCF7, MDA MB
231, and HEK293T cells were obtained from American Type Culture Collection,
ATCC. MCF10A cells were cultured in high glucose DMEM-F12 (Gibco,
31330-038) supplemented with 5% horse serum (Gibco, 16050–122),
1% penicillin/streptomycin (Thermo-Fisher Scientific, 15140-122),
20 ng/mg EGF (Sigma, E9644), 0.5 μg/mg hydrocortisone (Sigma,
H0888), 100 ng/mL choleratoxin (Sigma, C8052), and 10 μg/mg
insulin (Sigma, I1882). MDA MB 231, MCF7, and HEK293T cell lines were
maintained in high glucose DMEM (Gibco, 41966-029) supplemented with
10% fetal bovine serum (FBS) (Gibco, 10270106) and 1% penicillin/streptomycin
(Gibco, 15140-122). All cell lines were maintained in a humidified
incubator with 5% CO₂ at 37 °C. Overexpression of
SEMA6D was performed by a lentiviral expression system. Viruses were
prepared, and infections were carried out as explained previously.^{17 (link)} pLX304-SEMA6D plasmid was obtained from Dharmacon
(OHS6085-213576758) (GenBank reference: BC150253). LacZ
in pLX304 (pLX304-LacZ) was a gift from William Hahn (Addgene plasmid
no. 42560).^{18 (link)} Stable cell lines were generated
by 2 μg/mL blasticidin (Santa Cruz, sc-204655A) selection. MCF10A
cells that have increased Notch activation upon overexpression of
Notch1 intracellular domain (NICD) were described previously.^{17 (link)}

Primary SMNs were isolated from the spinal cord anterior horn tissue of 80 newborn SD suckling rats as described. Briefly, the spinal cord anterior horn tissue was dissected, mechanically dissociated, and then digested for 30 min with 2 mg/ml papain (Worthington Biochemical, Lakewood, United States) and a little DNase I (Thermo Fisher, Waltham, United States) at 37°C. The cell suspension was harvested and seeded on poly-D-lysine-coated cell slides in six-well culture plates (Corning, NY, United States) at a density of 7 cells/cm² × 10⁵ cells/cm² and cultured for 4 h in high glucose DMEM/F12 (Gibco, NY, United States) containing 10% FBS (Gibco, NY, United States) at 37°C with 5% CO₂. To remove contaminating glial cells, the medium must be exchanged with specialized Neurobasal-A (Thermo Fisher, Waltham, United States) supplemented with 2% B27 (Gibco, NY, United States) and 2 mmol/L Glutamax (Thermo Fisher, Waltham, United States) after 4 h cell adhesion. The quality of spinal motor neurons was analyzed by immunostaining of β-Tubulin.

The human cell line HepG2 was obtained from a Cell Resource Center (Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China). HepG2 cells were cultured in high glucose-DMEM/F12 (#11965-092, Gibco, NY, USA) containing 10% fetal bovine serum (FBS) (#04-001-1ACS, Biological Industries, Israel) at 37 °C in a humidified atmosphere, with 5% CO₂. The cell lines were routinely subcultured every 2 or 3 days.