Problems with lipids

Wolfgang Schechinger diabetes at lrz.uni-muenchen.de
Fri Aug 2 16:11:10 EST 1996


imcfarlane at its.dundee.ac.uk (IAN MCFARLANE) wrote:

Hi Ian!

May I can give you some hints? Currently I`m working with isoprenoids
I want my cells to tackle with. In earlier times, I had to do with
sphingolipids and their application cells.
In general, there are some ways to put your lipids on your cells: 

1) Adsorbe them to Albumine (BSA, e.g) before adding them to the
cells.
2) With my sphingolipids, I had good results (in getting them intgo my
cells ;-{ ) with the following procedure: 
Mix 10mg Lipid and 10 mg Phosphatidylcholine by dissolving them in
e.g. Chloroform (a few ml should do it) evaroprate thorougly by a
stream of N2 or by a vacuum centrifuge. Dissolve in (e.g.) 1 ml of
EtOH. Inject into 5 ml of warm (50 degrees C) PBS through a narrow
needle (I used an insuline syringe). Dialyze into PBS if you think,
the Ethanol would make cells dizzy. 
3) Incubate cells with lipds in medium at 4 degrees C for 30 or 60 min
to load them with your stuff. The low temp will allow diffusion but
prevent metabolizing.
4) Usually, I didn't store the vesicles longer than 24h (I dialyzed
them overnight and appied them on the next morning)
This should work for gangliosides as well

On what scale do you want to prepare your lipids? There are standard
protocols like the Bligh and Dyer one you mentioned or the Folch
extraction (if I remember right, *this* is the one for gangliosides.
For extracting negatively charged lipids like POLYphospho-inositides,
make the soln 1M with HCl to force the phosphates into the lipohile
phase


If you have an idea how I might solubilize my isoprenoids (liquid,
uncharged, (almost) non polar), please give me a hint.

>Hi,
> I'm about start looking at the effects of lipids on my adherent cell cultures. 
>I have previously used cholesterol solublised in ethanol and sterile filtered. 
>I now want to use some other lipids but need a bit of advice on the following 
>points.

>1) What should I solublise phospholipids in ?
>2) What should I solublise gangliosides in ?
>3) I want to extract lipid from tissue samples, will a 
>        chloroform/methanol ie Bligh-Dyer extract
>        all the lipids including the ones mentioned above ?
>4) Also once I have extracted my lipids what solvent should
>        I use to solublise and filter them ?
>5) For the above it would be easy to sonicate to form a micellar
>        suspension but how do you sterilise afterwards and if such a 
>         suspension is made hao long will it keep for ?

>If you can help me on any of the above points or suggest references 
> that might I would be most grateful

>Ian Mc





More information about the Methods mailing list