Can you heat RPMI 1640?

Kamalian, Laleh via methods%40net.bio.net (by L.Kamalian from liverpool.ac.uk)
Thu Feb 7 07:39:37 EST 2008


Hi
Thanks to everyone who answered my question regarding stable transfection of suspension cells.  As DK had suggested I found ready made semi viscous media for suspension cells and bought it.  However my cells are not growing in it, probably because the basic media is not the media that my cells are used to (RPMI 1640).  I had suspected this before using it but wanted to give it a try.  Now I want to make this using methylcellulose in my media.  I have found the concentration of the components and every thing else.  The only problem is how to dissolve methylcellulose in the media.  I found out that I have to add warm (60 ' C) liquid to the powder and let it cool down while stirring.  But can I heat RPMI 1640 media to 60"?  I couldn't find the answer on the net.
Thanks 
Laleh
 

________________________________

From: methods-bounces from oat.bio.indiana.edu on behalf of DK
Sent: Sat 19/01/2008 22:38
To: methods from magpie.bio.indiana.edu
Subject: Re: Stable transfection in suspension cells



In article <mailman.234.1200769898.2451.methods from net.bio.net>, "Kamalian, Laleh" <L.Kamalian from liverpool.ac.uk> wrote:
>Hi to everyone.  I am a PhD student and new to this mail list.  I have a
>problem I would like to ask.  I am working with cells which grow in
>suspension.  I have managed to get a good nock down in transient
>transfection using a plasmid vector and Lipofectamine as the transfection
>reagent.  Now I am ready to perform the stable transfection.  However I
>haven't got a clue how I will be able to separate the dead cells from
>the live ones in the selection process especially after the massive death
>happens.  Furthermore, I don't know how I'm supposed to pick a single
>clone even if I manage to get the live ones.  No one in our lab has ever
>transfected cells in suspension.  The selection method I will use is
>antibody selection (G418) for which I have  already started the optimization
>experiment in order to obtain the killing curve.  I was wondering if any of
>you have worked with suspension cells. Could you please give me some
>help?  Thanks
>Laleh.

This problem isn't new and there are several solutions.

1. Plate and select cells in 24-96 well format. For each well that gives
you survivors and massive cell growth, purify one individual clone by
limited dilution. (Google it).

2. Grow cells in viscous medium, so that clones stay together and,
more or less, stay apart from each other. For mammalian cells a
classic way to do that is carboxymethylcellulose mixed into medium.
(Can't remember concentration but you should be able to google it -
it's a standard thing in viral research). Then you just pick a clone with
pipet tip under the miscoscope, expand and, to be sure it's clonal,
purify by limited dilution.

3. If your cells survive overlay with a low-melting agarose, that's
a convenient option. Overlay with medium containing G418 and
1% LM agarose 24-48 hours posttransfection. Once the clone is
big enough, you just draw a circle around it and pick it with a tip
or Paster pipette. This is routinely done for insect cells/baculovirus.
The only drawback is that you need 2X concentrated medium
since, obviously, you don't want to autoclave your medium.

DK

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