*2.2. Sf9 Insect Cell Line, Culture Conditions and Baculovirus Infection*

The suspension-adapted lepidopteran insect cell line used in this work is the *Spodoptera frugiperda* (Sf9, cat. num. 71104, Merck, Darmstadt, Germany) gently provided by Dr. Berrow (Institute of Biomedical Research, Barcelona, Spain). Sf9 cells were cultured in Sf900III medium (Thermo Fisher Scientific, Grand Island, NY, USA) in 125-mL disposable polycarbonate Erlenmeyer flasks [25]. Cell cultures were shaken at 130 rpm using an orbital shaker (Stuart, Stone, United Kingdom) and maintained at 27 ◦C. HIV-1 Gag-eGFP VLP production was achieved through infection with the recombinant baculovirus *Autographa californica* multiple nucleopolyhedrovirus (*Ac*MNPV) (BD Biosciences, San José, CA, USA) enconding for the Gag-eGFP protein. Shortly, Sf9 cells were grown to 2–3 <sup>×</sup> 106 cells/mL and were infected at a multiplicity of infection (MOI) of 1. HIV-1 Gag-eGFP VLPs were harvested at 40 or 72 h post infection (hpi) by centrifugation at 1000× *g* during 15 min, and supernatants were kept at 4 ◦C until analysis. Non-infected negative controls reproducing cell growth conditions were also produced, for comparison.

Biophysical analyses were performed on HIV-1 Gag-eGFP VLP supernatants obtained as previously described. FreeStyle and Sf900III cell culture media and conditioned cell culture media obtained from HEK 293 and Sf9 non-transfected/infected conditions were also assessed in each analysis as negative controls.
