Unless otherwise specified, insects were incubated for 20 min, at 37 °C. After the incubation time, two different protocols were followed. For protocol one, to test the effects of CTX, CTB, and CTA on hemocyte concentrations in the insect Olaparib order hemocoel; hemolymph

(10 μl) was removed from the insect and the concentration of circulating hemocytes determined on a hemocytometer by phase contrast microscopy. Protocol two assessed the adhesive properties of the circulating hemocytes from insects injected with CTX, or its individual moieties. Hemolymph (10 μl) from these injected insects was added to slides (145 mm2) containing PBS (90 μl). Slides were shaken on a horizontal gyratory shaker (50 rpm, 30 min at 37 °C, ∼95% RH). The total hemocyte number and type attached to glass were determined by phase contrast microscopy. The data collected from protocol two were standardized by making a ratio comparing the absolute number of circulating hemocytes (in protocol 1) to the absolute number of attached hemocytes (in protocol 2). Where applicable, four fields per

slide area were counted for every replicate. Each of 5 insects received injections from individually prepared treatments (thus, n≥5 per experiment); an approach used in all subsequent experiments. To determine the effects of CTX and the stoichiometric concentrations of its moieties on removal of B. subtilis from the hemolymph, chilled larvae were co-injected with the chemicals and B. subtilis (6×107 cells in 10 μl PBS) and at the base of the third prothoracic leg. CAL-101 datasheet Insects were incubated for designated times on diet at 37 °C and bled after which bacterial numbers in the hemolymph were determined with a hemocytometer using phase contrast microscopy. In order to examine the effects of CTX on nodule formation, chilled insects (10 min on ice) were

swabbed with 70% (v/v) alcohol at the site of injection. Ten μl treatment solution (CTX) or control buffer was injected at the third prothoracic leg and the larvae incubated for 24 h at 37 °C on regular diet. Insects were monitored for normal behavior and mortality in order to preclude effects of physiological concentrations of the toxin on hemocyte behavior. Larvae were chilled on ice for 10 min. The in vivo hemocyte reaction was arrested by injecting the insect with 4% formaldehyde 6-phosphogluconolactonase (50 μl; v/v in PBS) with subsequent incubation on ice for 10 min. Insects were bisected ventrally and the frequency of melanized nodules determined with a stereo dissecting microscope. The number of replicates used was 20 larvae per experiment. Each larva was injected with independently produced bacterial-chemical treatments. Hemocytes from larvae were pooled in PBS as previously stated. One hundred μl of hemocyte suspension (∼2.4×105 cells) was added to polypropylene tubes (0.5 ml) containing 100 μl PBS, CTX, CTB, or CTA, and incubated vertically on a horizontal gyratory shaker (30 min, 37 °C, 200 rpm).