For ADCI assays, blood stage parasite cultures were synchronized by at least two successive sorbitol treatments followed, after maturation over 24 h, by floatation on 1% porcine skin gelatin type A (Sigma).
F1 larvae resulting from the jumpstarter-UAS-eGFP reporter cross were flushed from media with water, cleared of debris by floatation on NaCl solution, rinsed in water, and screened for fat body GFP expression under a fluorescence dissecting microsope using a GFP filter.
The relative intensity of hookworm infection, in eggs per gram, was determined by floatation, using a sodium nitrate solution (specific gravity 1.20) (23 ).
After 12 hours, the larvae were collected by floatation in a 20% glucose solution, rinsed with phosphate buffered saline (PBS), and transferred again, either to a fresh plate with glucose fly food or to a fresh plate with fly food containing 555 mM glucose plus 225 mM galactose.
To investigate whether the active siRISC also physically associates with membranes, post-nuclear lysates of HeLa cells transfected with SSB siRNA were subjected to a membrane floatation assay on a discontinuous sucrose gradient (Tahbaz et al, 2004).
The reason that air bubbles have a rounded shape when very small and a flat-bottomed shape when large is that interfacial energy will have a measurable influence on small bubbles, but is overtaken by floatation forces for larger bubbles.
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Consequently, the preferential partitioning of SP-predicted proteins within the cell wall and endomembrane components specifically enriched in the R-fraction suggested that DIMs containing proteins targeted to secretion might not have a buoyancy similar to those uniquely retrieved by floatation (e.g., mainly PM and plasmodesmata DIMs).
After 12 hours on the new food, the larvae were again collected by floatation and stored at −20°C until the RNA was extracted using the RNeasy Mini Kit (Qiagen) as recommended by the manufacturer, with DNase digestion performed on the column.
