Empty mobicol spin column

Adult haemolymph was collected as follows. Fifteen individuals were placed on a 10 µM filter of an empty mobicol spin column (MOBITEC), covered with glass beads and centrifuged for 20 min at 4°C at 2169 g. Haemolymph was recovered in 50 µl protease inhibitor solution (Roche; one tablet dissolved in 4 ml PBS) and protein concentrations adjusted after a Bradford test. Sample volumes were adjusted to 200 µl in 5 mM CaCl₂ solution (diluted in protease inhibitor solution, see above) and after addition of 800 µl of L-DOPA solution [20 mM in phosphate buffer (pH 6.6)] the samples were incubated at 29°C in the dark. After 30 min, the optical density at 492 nm was measured for each sample against a L-DOPA control containing no haemolymph. As activation of the proPO system was blocked by the presence of the protease inhibitor, the values reflect the in vivo PO activity at the time of infection. Melanization assays were repeated ten times.

Larval and adult hemolymph was collected as follows. Fifteen to twenty individuals were placed on a 10 µM filter of an empty mobicol spin column (MOBITEC), covered with glass beads and centrifuged for 20 minutes at 4°C, 10'000 r.pm. Hemolymph was recovered in 50 µl protease inhibitor solution (Roche; one tablet dissolved in 4 ml phosphate-buffered saline, PBS) and protein concentrations adjusted after a Bradford test. Sample volumes were adjusted to 200 µl in 5 mM CaCl₂ solution (diluted in protease inhibitor solution, see above) and after addition of 800 µl of L-DOPA solution (20 mM in phosphate buffer pH 6.6) the samples were incubated at 29 °C in the dark. After 30 min, the optical density at 492 nm was measured for each sample against an L-DOPA control containing no hemolymph. Since activation of the proPO system was blocked by the presence of the protease inhibitor, the values reflect the in vivo PO activity at the time of wounding. Each experiment was repeated three times.