0.45 μm filter units

Lentiviruses were produced in 293FT cells using Lipofectamine 2000 (Invitrogen) transfection reagent. Supernatants of 293FT transfectants were collected 48 hours after transfection and filtered using 0.45 μm filter units (Sartorius). JP5 cells were grown to 50–60% confluency and transduced with lentiviral particles in the presence of hexadimethrine bromide (10 μg/ml Polybrene, Sigma-Aldrich). Cells stably expressing firefly luciferase (LUC2) or human gremlin-1 were obtained by selection with hygromycin (200 μg/ml) or puromycine (20 μg/ml), respectively. Cells expressing LUC2 were further transduced with lentiviral particles containing either pLVX-puro empty vector or pLVX-puro carrying human gremlin-1. Western blotting against gremlin-1 was performed to assess the overexpression efficiency.

The processes of YFV production and purification are similar to those described by Pato and coworkers [10 (link)]. The working virus seed of YF 17D-213 infects the Vero cells (WHO-approved cell line) and undergoes bioreactor culturing. The supernatant of the culture medium containing the virus was treated with β-propriolactone for 24 h at room temperature to inactivate virus, and then concentrated 100 times by using a molecular weight cutoff of 300 kDa membrane filter (Sartorius, Germany), followed by downstream chromatography purification. Chromatography was performed using Akta pilot, operated with the UNICORN software (https://www.cytivalifesciences.com/en/us/shop/chromatography/software/unicorn-7-p-05649) (Cytiva, Marlborough, MA, USA). Ion exchange (Q Sepharose Fast Flow) and Capto Core700 (Cytiva, USA) resin prepacked in an XK50 Column were used for capturing and polishing, respectively, and the entire purification process. The purified sample was subsequently filtered using 0.45 μm filter units (Sartorius, Goettingen, Germany).