Leibovitz s l 15 medium

The TiLV inactivated vaccines were prepared as described previously by Mai et al. (2021). Briefly, TiLV strain TH-2018-K, which was isolated from Nile tilapia during a TiLV outbreak in Thailand in 2018 [20 (link)], was propagated on E11 cells, in Leibovitz’s L15 medium (Sigma, Saint Louis, MO, USA) containing 5% fetal bovine serum, until a cytopathic effect (CPE) of approximately 80% of the cell monolayer was achieved. The supernatant containing the virus was collected and clarified to remove cell debris, by centrifuging at 4500× g for 5 min at 4 °C. Virus concentration was determined using 50% tissue culture infectious dose (TCID₅₀/mL) [30 (link)]. The virus was inactivated by either heating at 60 °C for 2.5 h or incubating in a 0.006% formalin solution (16.2 µL formalin 0.37% in 1X phosphate-buffered saline [1× PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄ and 1.8 mM KH₂PO₄, pH 7.4] per 1.0 mL viral stock) at 25 °C for 24 h. Viral infectivity was tested on E11 cells and successful inactivation was confirmed when no CPE was observed after 7 days. The vaccines were stored at 4 °C until used. The virus concentration was determined to be 1.8 × 10⁷ TCID₅₀ per mL before being used. All chemicals used were purchased from Merck (Kenilworth, NJ, USA).

MCF 10A (non-tumorigenic), MCF7 (ESR + BC), and MDA-MB-231 (triple-negative phenotype) breast cells lines were obtained from ATCC and cultured with Dulbecco’s Modified Eagle Medium (DMEM) (Cultilab), Iscove’s Modified Dulbecco’s Medium (IMDM) (Cultilab), and Leibovitz’s L-15 Medium (Sigma Aldrich), respectively. MCF 10A was supplemented with 10% of fetal bovine serum (FBS), 20 ng/mL of epidermal growth factor (EGF), 500 ng/mL of hydrocortisone, 10 µg/mL of insulin, and 50 μg/L of gentamycin. The malignant lineages were supplemented with 10% of FBS and 50 μg/L of gentamycin. All lineages were incubated at 37 °C in a humidified atmosphere containing 5% CO₂ atmosphere until they reached 80% of confluence.

For protein mobility measurements inside and outside Gag assembly sites, 3 × 10⁵ Jurkat T-cells were suspended in in 200 mL Leibovitz’s L-15 medium (Sigma-Aldrich) 72 h post-infection with NL4.3 HIV-1 Gag.iGPF. Infected cells were stained for Env in suspension at 16 °C by incubation with 2G12 Fab fragments and anti-human Abberior STAR RED (KK114) conjugated Fab fragments for 1 h each in 0.5% BSA/L-15 medium. Cells were then washed three times in L-15 medium, resuspended in 250 mL of L-15 medium and adhered to solution poly-L-lysine (0.1 mg/mL) (Sigma-Aldrich)-coated glass surface of eight-well Ibidi m-Slides (Ibidi, Gräfelfing, Germany) at 37 °C 30 min prior to microscopy measurements.