Surface staining was performed after ALDH1 reaction, while keeping the cells in the staining buffer on ice to prevent pumping BODIPY-aminoacetate out by ABC transporters.
The DNA samples were then precipitated, heated at 90 °C for 4 min in loading denaturing buffer, and chilled in ice prior to being loaded on an 8% denaturing polyacrylamide gel for 90 min at 65 W in TBE buffer.
The cells were washed, counted and resuspended in FACS buffer for 30 min on ice to block non-specific staining.
500 μg of midguts were centrifuged 5 min at 12,000 g to discard the old buffer, resuspended in ice-cold fresh MET buffer containing 1 mM PMSF and homogenized with 30 strokes of a glass-teflon homogenizer.
Cells were washed once with PBS buffer, resuspended in ice-cold mitoprep buffer (0.225 mol/L manifold, 0.075 mol/L sucrose, and 20 mmol/L HEPES pH 7.4), and broken in a glass-Teflon homogenizer on ice with 30 strokes.
Cells were washed with PBS buffer, fixed in ice-cold 75% (vol/vol) ethanol, and then stained with 2% (wt/vol) Alizarin red S (Sigma-Aldrich).
Cell pellets were re-suspended in 50 μl of cold lysis buffer and incubated in ice for 15 min.
E. granulosus adult worms were washed in PBS, suspended in lysate buffer, and sonicated in ice until the suspension became clear.
In order to obtain the most dilute hemoglobin concentrations, the packed cells were treated with large amounts of hypotonic buffer and kept in ice for 20 minutes.
Tissue samples were homogenized in hexadecyltrimethylammonium bromide buffer in a glass homogenizer on ice.
