Stable Cell lines

mark hill m.hill at unsw.edu.au
Thu Nov 12 03:08:09 EST 1998


You are Joking? The islands of cells are your clones. You need to autoclave
some vacuum grease, chop the lids and tapered bases off some eppies,
autoclave them.

Draw circles around your "islands" form below. In the laminar hood use
sterile forcepsdip the top end of your eppies in the grease, place them over
your makred "islands" then trypsinize the cells off with 1-2 drops trypsin
1min. Add your media and replate into 24 well dishes, an "island to a well".
Have fun

Sailesh Surapureddi wrote:

> My Dear Expressionists
>
>         I am trying to make some stable cell lines. I have tranfected my
> constructs (has amp and neo as markers) to fibroblasts (Ca-Phos method).
> After the removal of calcium precipitate, cells were split and seeded in
> low density and treated with Neomycin sultfate (500 microgram/ml). The
> control cells after sometime die out. But intrestingly all the lines which
> I am trying to develop have confluent cells but the intresting thing is
> that there are lot of
> "islets of cells" with clear demarcations (they look kinda dark in color
> when compared to the other cells in normal microscope observations.)
>
> My Q is there an kind soul who knows what I am talking about or observed
> such islets when making stable cell line ?
>
> 2. Can anyone suggest me on how to pick up individual islets without
> disturbing the rest (I can only see them under microscope and not
> distinguiable to normal vision)???
>
> All your help is greatly appreciated in advance
>
> Sailesh.
>
> Sailesh Surapureddi, Ph.D
>                                 Communicators
> Plan #12, Cellbiologi           Work    +46-13-223917
> Dept. of Biomedicine & Surgery  Res     +46-13-274718
> Linköpings University           Cel     +46-70-4916570
> S 581 85, Linkoping             Fax     +46-13-224149
> Sweden.                         Email  Saisu at mcb.liu.se

--
___________________________________________________________
Dr Mark A. Hill                               Email: m.hill at unsw.edu.au
Cell Biology Lab                              Ph: +612 9385 2477 (office)
School of Anatomy                            Ph: +612 9385 2474 (lab)
University of NSW  2052                   Fax: +612 9313 6252
Sydney, AUSTRALIA                     http://anatomy.med.unsw.edu.au/cbl





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