Stable transfection in suspension cells

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 havealready started the optimization experiment in order to obtain the killingcurve. I was wondering if any of you have worked with suspension cells. Could you please give me some help? Thanks
Laleh.

Dear Laleh,

you could try this: make a dilution of your cells that you have 5-10
cells per ml. Then seed 100ul in each well of a microplate (a total up
2-5 plates) under selection conditions. You'll get approx 1 cell per
well. Then watch them become more (or not). You might use a medium
containing 50% conditioned medium from 293 HEK (or similar well
growing cells), just take the not too much used medium and filter
through a syringe filter in order to remove any cells. Then you cells
they don't feel so lonely, means they get some growth factors and grow
more easily. Then expand those wells where your cells grow (into 24
well plates) and check aliquots for gene expression. You might re-
check your cells for expression from time to time, as there is some
chance that a) those loosing the transgene overgrow your culture and
b) that you had more than 1 cell in the initial microwell.

If you have access to a cell sorter (FACS), then apply selection
pressure after transfection and collect the propidium iodide negative
cells (adding Ethidium bromide 10mg/ml stock 1:10.000 might do it as
well) for microplate seeding. This probably makes things much easier,
especially when you get a low transfection rate.

Good luck!

Wo

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: [Only registered users see links. ] on behalf of DK
Sent: Sat 19/01/2008 22:38
To: [Only registered users see links. ]
Subject: Re: Stable transfection in suspension cells



In article <[Only registered users see links. ].net >, "Kamalian, Laleh" <[Only registered users see links. ].uk> wrote:

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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In article <[Only registered users see links. ].net >, "Kamalian, Laleh" <[Only registered users see links. ].uk> wrote:

Yes, absolutely, you can. Wouldn't be a good idea to do it for a very
long time but all normal media will tolerate 60C without obvious
degadation of glutamine. If you are truly paranoid, you can add
1-2 mM of Glutamax (Ala-Gln peptide that is heat stable and is
hydrolysed in the cells to make Gln).

DK

Am 07.02.2008, 08:39 Uhr, schrieb Kamalian, Laleh
<[Only registered users see links. ].uk>:


I would produce a 10 or 20 x stock solution of Methylcellulose in water
and autoclave to get it sterile. Then use 10 x RPMI (commercially
available or prepared from powder) to make the final medium.

[lizotova]

Stable transfection in suspension cell

[jackquality]

Hi

You can see same topic at the side bar of this site. You can find out some thing same your questions.

Or you can see more at: [Only registered users see links. ]

Rgs

[jubileelin]

Dear Laleh,

I am a graduate student and a new one in the station. I am doing stable clone transfection in suspension cells too. After searching google, this site gave most helpful information. So I decide to use the same method as your choice. Could you give me more details about how to use methylcellulose media to select the stable clone? Thank you so much. Please email me at [Only registered users see links. ].

Linda

[jjsimz]

Hi! I am also a graduate student doing colony picking from a stable transfection. Could you send the protocol to me too? Thanks a lot!

[Only registered users see links. ]

[ssongmen]

Hi
I am also a new PhD student and new to this forum. I am also trying to establish stable cell line of K562 suspension cell. And I want to know the protocol to use carboxymethylcellulose for identification of clone after transfection. Could anyone Laleh or jjsimz or Linda forward it to me.

Thanking you in advance.

My email is [Only registered users see links. ]