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cells washed away - cell culture problems (Mar/14/2002 )

Hello everybody,

I'm working with a LbetaT2 cell line. I have to do stimulations with GnRH daily. So, I have to rinse, to add medium, to sample and to rinse and add medium again. One big problem is, that after one week, 25% of all the cells are washed away, resulting in wrong results. I read in a couple of articles that some people use thermanox coverslips, coated with Matrigel. Is the enhancing the adherence of the cells or how can I improve the adherence.

>With kindly greetings,

-S70-

sounds like ur cells do have some problems remaining attached to the substratum.

collagen coating previous to the plating the cells have helped soem ppl.  maybe u should look into that and yes. tha't what matrigel is for( aiding celll adehsion to the substratum. well others use matrigel for othe thigns too but in that case u mentioned.... )

-sean-

Hello Sean,

Thank you for the information. I'll try this way and I will let you know what the results are. The other manner of using Matrigel is less scientific I suppose?

With kindly regards,

Frank Eertmans

-S70-

hey frank

well i didnt meantobelittle the use of matrigels. they are quite useful when working certain types of cells. ( as those that don;t attach to subtratum well.)  if u dont have matrigel, u can also try collagen coating( or lamininor fibronectin etc note these are ECM proteins)

there r some other uses of matrigel such as activation of various sig. cacade by singallign thorugh RTK's as well.  

gd luck with ur exp's.

-sean-

Hello Sean,

Thank you very much for the information. I ordered Matrigel, so I hope that it will be better now!

CU

Frank

-S70-

Some thoughts on the use of Matrigel (from a different Sean):

1) its expensive, you could probably produce a ECM coated surface using collagen, (choose the type based on the cell type you are using), fibronectin or laminin. Also note that FBS contains a large amount of fibronectin and vitronectin, so you might even be able to coat with serum !

2) ┬áMatrigel contains more than just ECM proteins, it contains growth factors (Exp Cell Res 1992 Sep;202(1):1-8) including TGF-├č, EGF, bFGF, PDGF and IGF.

Just so you are aware.

-Sean Peel-

Hello Sean,

Thank you for the information. I know Matrigel is quite expensive. But now, I allready tried Poly-D-Lysine, Collagen Type I and a 0.1% Gelatin Solution, but my cells (pituitary gland) don't even attach to the bottom of my plate. They do when I just plate them out in non treated plates. I used the protocol of the manufacterer to coat them. In the little amount of available literature, concerning my cells, they use Matrigel. Maybe it's the only thing that works. Anyway, thank you very much for the information.

With kindly regards,

Frank Eertmans

-S70-