Gibco grace s medium 1

Spodoptera frugiperda derived Sf21 cells were cultured at 25°C in 25 cm² tissue culture flasks (T25 flasks, Nunc) containing 4 mL of Gibco^®Grace’s Medium (1×) (Life Technologies™, Paisley, UK) supplemented with 10% heat-inactivated fetal bovine serum (FBS). Sf21 cells stably expressing the wild type SeABCC2 gene, designated as Sf21-FRA [37 (link)], were maintained at 27°C in the same culture medium supplemented with 10 μg/mL Blasticidin.

Cultured Sf21 insect cells, originally derived from S. frugiperda, were maintained in 25 cm² tissue culture flasks (Nunc T25 flasks, Thermo Scientific™, Waltham, MA, USA) at 25 °C with 4 mL of Gibco^® Grace’s Medium (1×) (Life Technologies™, Paisley, UK) supplemented with 10% heat-inactivated fetal bovine serum (FBS).
For transient expression, cells were seeded on 12-well plates with the same medium without FBS at ca. 70% confluency and transfected with 0.5 µg of the pIZT/V5His/HvmALP1 or pIZT/V5His plasmid using Cellfectin^® II Reagent (Thermo Scientific™, Waltham, MA, USA), following manufacturer’s instructions. Five hours post-transfection, the medium was replaced with fresh medium containing 10% FBS. After 24 h, cells were examined using a confocal microscope (Olympus, FV1000MPE, Tokyo, Japan) equipped with the appropriate filter for green fluorescent protein (GFP) detection as transfection marker. The enzymatic activity of alkaline phosphatase was then measured as explained above.