| Condition |
200 uL of agar (2 %) was deposited into each well of a 96-well microtitre plate. After this had solidified, nematode egg solution (30 uL) was added into each well. Water (10 uL) was added to the control wells and cyclotide solution (10 uL, varying concentrations) was added into the treatment wells. The final concentration range was 0.3-33.3 ug mL-1 (0.09-11.53 uM). After 24 h, growth medium (20 uL) was added to each well. The growth medium consisted of Earles salt solution (10 %, v/v), yeast extract (1 %, w/v), sodium bicarbonate (1 mM) and saline solution (0.9 % sodium chloride, w/v). The nematodes were allowed to feed and develop for four days and then killed using Lugols iodine solution and scored for the number of fully developed infective stage larvae (L3) present in each well. Each treatment was conducted in at least triplicate and controls for water and/or ethanol (20 %) were included in each assay as required. |
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