Opti memtm 1 reduced serum media

HEK293T cells expressing S-M1 VLPs (HEK293T-S-M1) were grown in Opti-MEM^TM I reduced serum media (Thermofisher Scientific) in tissue-treated 15-cm² dishes (5 dishes per production) for 72 h. The cell supernatant (up to 100 mL) was collected and concentrated through a 50 kDa Amicon^® Ultra Centrifugal Filter Unit (Merck, Darmstadt, Germany) (final volume of 25 mL). The concentrated sample was subjected to ultracentrifugation through a 20% glycerol cushion at 135,000× g, 4 °C for 4 h. Cell pellet containing S-M1 VLPs was harvested and re-suspended with 500 µL of phosphate buffer saline (PBS). The S-M1 VLPs were quantified by dot-blot analysis against rabbit α-spike antibodies. The purified spike protein expressed in HEK293T cells was used as a standard.

Human embryonic kidney (HEK) 293T cells were purchased from ATCC^® (CRL-3216); ATCC^®, Manassas, VA, USA). Cells were maintained in Opti-MEM^TM I reduced serum media (Thermofisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Thermofisher Scientific, Waltham, MA, USA). Vero E6 cells were cultured in EMEM containing 2% FBS, 1% L-glutamine, 1% penicillin-streptomycin (Thermofisher Scientific), 40 µg/mL gentamicin (Thermofisher Scientific), and 0.25 µg/mL fungizone (Thermofisher Scientific). All cells were maintained in a 5% CO₂ incubator at 37 °C.