Penicillin streptomycin and l glutamine

Cells were cultured at 37 °C in 5% CO₂. Human BE(2)-M17 neuroblastoma cells (called M17D, ATCC number CRL-2267) were cultured in DMEM/F12 GlutaMAX (Thermo Fisher Scientific), 10% fetal bovine serum (Sigma) and 1% penicillin/streptomycin. Prior to the transfection, cells were seeded at a density of 1.5 × 106 cells/6 cm dish. HEK cells were cultured in DMEM with 10% fetal bovine serum (Thermo Fisher Scientific) and 1% penicillin/streptomycin and l-glutamine (2 mM, Gibco). Transfections with αS wild type and fPD mutants (A30P, E46K, G51D, A53T, H50Q) were carried out using Lipofectamine 2000 according to the manufacturer’s instructions. Cells were harvested 48 h after transfection, pelleted, snap frozen and further processed by cross-linking. HEK293 αS C-term Strep II-tagged lysates were purchased from GeneScript.

Sera were heat inactivated at 56°C for 30 minutes and tested for nAb to the MP-12 virus as described previously.²⁰ (link) Briefly, equal volumes of 2-fold dilutions ranging from 1:5 or 1:10 through 1:1280 of each sample were prepared in minimal essential medium and incubated overnight at 4°C with an equal volume of 50 to 100 PFUs of the MP-12 virus. On the following day, 50 µL of the virus–sera mixture were inoculated in duplicate onto a confluent monolayer of Vero E6 cells grown in 24-well plates. Cultures and inocula were incubated for 1 h at 37°C and 5% CO₂. A mixture of 1% SeaKem agarose (VWR) with an equal volume of 2× Eagle’s basal medium with Earle’s salt, HEPES, sodium bicarbonate, 8% fetal bovine serum, and 1% penicillin/streptomycin and L-glutamine (Thermo Fisher Scientific) was prepared and 0.5 mL was overlaid onto each cell culture. The agarose overlay was allowed to solidify, and then the cultures were incubated for 3 days at 37°C and 5% CO₂. At 3 days post-infection, cultures were stained with a 0.33% neutral red solution and incubated for 4 to 6 hrs to identify PFUs. The PFUs were counted and the dilution of serum that reduced the number of plaques relative to the negative human serum control by 80% was considered to be the nAb titer.