At the end of the incubation (see above), the culture medium was removed
and the cells were washed with phosphate buffered saline (PBS)
before being detached using 0.05% (w/v) trypsin/0.02% (w/v) ethylenediaminetetraacetic
acid (EDTA) in PBS. The detached cells were pelleted
by centrifugation and then washed with 1% (w/v) FAF-BSA in HBSS to
remove any residual [1-14C]-labelled fatty acids. Total lipid was extracted
as described above and the different classes of lipids were separated
on HPTLC plates developed in methyl acetate/isopropanol/chloroform/
methanol/0.25% (w/v) aqueous KCl (25/25/25/10/9, by volume) as
described previously (Vitello and Zanetta, 1978). The separated lipids
were visualised by placing the plates in a tank saturated with iodine
vapour. The areas of silica gel corresponding to the different classes of
lipids were scraped from the plate into separate vials and radioactivity
was assayed using a TRI-CARB 2000CA scintillation counter (United
Technologies Packard, Pangbourne, UK). The transformed spectral
index of external standard (tSIE) value was used as a measure of
quenching. This value was used to calculate the counting efficiency
according to the equation, efficiency = (tSIE × 0.033) + 56 based on
a quenching calibration curve. This efficiency value was then used to
convert counts per minute (cpm) to disintegrations per minute (dpm)
using the equation dpm = (cpm / efficiency) × 100.