Preparation of delipidated FCS (Thiele lab)

From LipidomicsWiki

Preparation of delipidated FCS (Thiele lab)

Rationale: This procedure gives fetal calf serum that is free of cholesterol, triglycerides and most other lipids.
Protein is not removed. This FCS can be used to grow cells under defined lipid conditions, or to obtain cells
with very low amounts of stored neutral lipids. Most cell lines tolerate growth in this serum for at least 3 days.

-    Whole procedure takes 4 days
-    defrosting 0,5 l FCS takes 1h
-    use glass pipets only
-    use always gloves when handling dialysis membrane
-    cool 13 l 1 x PBS to 4 degrees Celsius for each day

step 1    for 500 ml of FCS prepare a mix of:
            - 400 ml Diisopropylether (Sigma 24241 or other)
            - 200 ml n-Butanol (AppliChem A0771,2500 or other)
step 2    on a magnetic stirrer, intensely stir the solvent mixture with 500 ml FCS for 30 min
step 3    centrifuge at 4000 g until phases separate (15 min.)
step 4    discard upper phase
            - transfer the lower phase into a fresh beaker, using glass pipets
step 5    stir lower phase with 500 ml Diisopropylether for 30 min
step 6    let phases separate
            - use separating funnel, takes at least 2 h
            - centrifugation also possible (see step 3)
step 7    remove upper phase completely, using the separating funnel or pipetting out the lower phase (see step 4)
step 8    under gentle stirring, blow a stream of nitrogen over the surface of the lower phase for 2 h
step 9    dialyze against 13 l (or more) of 1 x PBS three times at 4 °C (over night)
            - dialysis membrane: Spectra/Por MWCO 12-14,000 (Spectrum No. 132700)

step 10    filter-sterilize, aliquot (25 ml), store at -20 degrees Celsius