Class 2 type a b3

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.

A commercial sodium hypochlorite solution (6%; The Clorox Company) was diluted in sterile water at a ratio of 1:2 to reach a concentration of 20,000 ppm. Purchased alfalfa and fenugreek seeds (2 g) were treated with the diluted sodium hypochlorite (5 mL per gram of seeds) with agitation on a platform shaker (Model 421105, Nutator) for 15 min at room temperature. Spent sanitizer solution was aspirated, and residual sodium hypochlorite on alfalfa or fenugreek seeds was neutralized with 5 mL Dey-Engley neutralization broth (BD) for 10 min with agitation. The seeds were then rinsed twice with 5 mL of sterilized deionized water before being dried on a piece of sterilized weighing paper (Fisher Scientific) for 1 h in a biological safety cabinet (class II type A/B 3, Nuaire) at room temperature.