Heilbrigðisskýrslur - 01.12.1990, Blaðsíða 139

well as calcium and glucose (see above). Cells were spun again, the supernatant
discarded, and the remaining cells weighed and dispersed in 20 ml of
Waymouth's (MB 752/1) culture medium (Gibco) containing foetal-calf serum
(10%), and supplemented with penicillin (200 units ml*1), streptomycin (200 pg
ufi'1), glutamine (3.4 mM), serine (0.5 mM) and alanine (0.4 mM). Cells were
filtered again, counted in a haemtocytometer chamber with a Swift Instruments
Intemational microscope ( x 40 objective). The cells were suspended at a conc. of
0-70 - 0.90 x 10*6 cells ml*1 and plated out (3.0 ml per dish) on to Falcon ’Primaria’
culture dishes (Becton Dickinson Labware, Oxnard, CA, USA). The dishes were
placed in an incubator (Leec Automatic C02 Incubator) under an atmosphere of
air/C02 (19:1) (humidified) at 37° C. After 4 - 6 h, most of the cells had attached to
the base of the dishes, and the medium containing unattached cells was removed.
The remaining monolayer of cells was washed with phosphate-buffered saline
(PBS), and serum-free supplemented culture medium was added together with
Pyruvate (1 mM), lactate (10 mM), dexamethasone (1 p.M) and oleate (0.75 mM
bound to bovine serum albumin, 0.5%), and the various substances to be tested in
^ccordance with experimental protocols. The dishes were returned to the
'ncubator and the cells incubated for 24 h. In experiments that lasted more than
24 h, culture medium was changed every 24 h. The perfusion apparatus,
glassware and tools were autoclaved before use. The perfusion buffer was
sterilized by passage through Millex®-GS Filters (Millipore® S.A., Molsheim,
Prance) (pore size 0.22 (im).
Cell harvesting and analytical methods: At the end of the final 24 h of culture,
medium from the individual dishes was collected and, in order to remove^ cellular
debris, was spun for 20 min at 16,000 rpm (= 21,000 g) in a MSE HI-SPIN-21
centrifuge, using an 8 x 50 ml fixed-angle rotor (cat. no. 43114-143). The
supernatant was placed in 13 x 64 mm Beckman polyallomer Bell-Top Quick-Seal
lubes, each containing 150 pl of the following „perservative cocktail" (NaEDTA,
4-4 g, chloramphenicol, 0.293 g, NaAzidie, 0.367 g, gentamycinsulphate, 0.293,
Trasylol®, 36,667 units, in 100 ml of distilled water). The tubes were sealed and
centrifuged at 40,000 rpm ( = 145,000 g) for 16 h in a Beckman L8-70 ultracentrifuge
with a 44-place 50.4 Ti fixed-angle rotor. The floating fraction of d<1.006 g.ml1
was obtained by tube slicing. A portion of the VLDL-containing fraction was used
l°r extraction of total lipids [8]. The total lipid fraction was dissolved in ethanol
(Ö-4 ml), and the total mass of triacylglycerol and cholesterol (non-esterified and
esterified) was measured by enzymatic colourimetric methods, using kits
Purchased from Boehringer ('Triglycerides GPO-PAP method' and 'Cholesterol C
System, CHOD-PAP method', respectively). Manipulative losses of
triacylglycerol were accounted for by addition of glycerol 14C-trioleate as an
lr*ternal standard. After removal of the medium, ice-cold phosphate-buffered
saline (PBS) was added (2 ml per dish), the cells were detached from the plates with
u rubber “policeman," and the material was transferred to a centrifuge tube. Each
lsh was washed with another 2.0 ml of PBS, and after combination, the cells were
°btained by centrifugation at 80 g for 10 min. The supernatant was removed, and
!he cells were sonicated (MSE M-12 sonicator) for 10 s at 8 (im (peak-to-peak) after
^uspension in 0.5 ml of PBS. A sample of the sonicated material was used for
etermination of total protein [9]. The rest of the sample was used for estimation of
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