Sterile water for injection

Neutralization IC₅₀ values were determined using PRNAs. First, MDCK cells were seeded at 8 × 10⁵ cells/ml onto 12-well plates. The following day, MAbs were diluted to 100 μg/ml in 300 μl of 1× MEM and then serially diluted 1:5 in a 24-well plate to a final concentration of 0.032 μg/ml in 1× MEM. Virus was diluted to 1 × 10³ PFU and added to each of the antibody dilutions (50 μl/well). The virus-MAb mixture was incubated at room temperature for 1 h, shaking. The MDCK cells were then washed one time with 1× PBS and immediately infected with 200 μl of the virus-MAb mixture and incubated at 33°C with 5% CO₂, with the plates rocked every 10 min. In the meantime, the overlay was prepared by diluting MAbs to 100 μg/ml in 625 μl of 2× MEM and then serially diluted 1:5. Then, a mixture of 1× DEAE-dextrane and 1 μg/ml TPCK-treated trypsin in sterile water for injection (Gibco) was added at 180 μl per well. After the 40 min, the inoculum was aspirated (three wells at a time) and immediately replaced by the overlay mixture containing 360 μl of 2% Oxoid agarose so that the MAb concentration within the agarose matched the same MAb concentration of the inoculum. The plates were incubated at 33°C with 5% CO₂ for 3 days and then fixed with 3.7% PFA overnight at 4°C. The overlay was removed, and the cells were stained as described above. PRNAs were conducted in duplicates.

Two days prior to honey bee pupal cell experiments, a brood frame containing purple-eyed pupae was brought into the lab. Fine-point, curved-tip forceps (Bioquip) were used to carefully remove the wax capping from pupae. Wide-tip, featherweight forceps (Bioquip) were used to gently grasp pupae between the eyes to remove them from the wax comb cells in which they develop. Pupae were incubated at 28 °C overnight in 12-well plates, and wounded (i.e., melanized) pupae were discarded. Primary cells were harvested from surface-sterilized honey bee pupae in a biosafety cabinet (Class II type A/B3, Nuaire). Surface sterilization was carried out in a sterile polystyrene petri dish (100 mm × 15 mm, Fisher), in which pupae were swirled in 0.6% hypochlorite solution (diluted bleach) for 3 min, 70% ethanol for 3 min, and briefly in sterile water for injection (Gibco). In groups of two, pupae were dissected into head, thorax, and abdomen segments in 2 mL WH2 medium in a 47 mm dish using sterile 18-gauge needles to vigorously disturb tissues and release cells into the medium [175 (link)]. The media containing the cells was then transferred to a 50 mL conical tube, and cells from individual pupa (~24 × 10⁶ cells) were pooled. Cell suspensions with approximately 10⁶ cells per 300 μL were plated into each well of a 48-well plate and incubated at room temperature overnight before infection.