In vitro spike S1 neutralization experiments of nanodecoys

Recombinant spike S1 (Sino Biological 40591-V08H, 10 ng/mL, MW = 76.5 kDa) was added to nanodecoys at different concentrations (5×10⁹, 1×10⁹, 2×10⁸, 4×10⁷, 8×10⁶, 1.6×10⁶, and 3.2×10⁵) and incubated for three hours. After that, the unbound spike S1 was removed by ultracentrifugation (100 kDa). Spike S1 before and after binding to nanodecoys was determined using an ELISA kit (Sino Biological SARS-CoV-2 SPIKE ELISA KIT, Sino Biological) according to manufacturer’s protocol. To study the neutralization of spike S1 with nanodecoys in primary lung derived cells (LSCs), spike S1 was first labeled using NHS-Rhodamine (46406, Thermo Fisher Scientific) according to the manufacturer’s instructions. The RhB-spike S1 (100 ng) was first incubated with LSCs (2×10⁴) in 4-well slides for 1 h and washed with PBS for three times. After that, DiD labeled nanodecoys (2×10⁷) were added and incubated for another 4 h. Cells were washed and fixed using 4% PFA prior to stain with Alexa Fluor^™ 488 Phalloidin (Invitrogen^™ A12379). Cells were imaged using an Olympus FLUOVIEW confocal microscope.