In vitro Assays for suspension cells

Dr Engelbert Buxbaum via methods%40net.bio.net (by engelbert_buxbaum from hotmail.com)
Tue Jun 24 14:05:43 EST 2008


Am 18.06.2008, 19:03 Uhr, schrieb Kamalian, Laleh  
<L.Kamalian from liverpool.ac.uk>:

> I am working on SCLC cell lines which grow in suspension.  I have had  
> lots of trouble so far as I am the only one in the lab working with this  
> kind of cells.  I would like to examine tumourogenisity, invasion and  
> proliferation ability of my parental malignant cells comparing to the  
> cells with reduced expression of specific genes.  So far the only assay  
> I have found to be straight forward is soft agar assay for  
> tumourigenesis.  Even that seems to be more of a proliferation  
> assessment than adherence-independent grow, but at least it is doable.

Normal cells (e.g. mouse fibroblasts) would not grow under the conditions  
of the assay. The better cells do grow, the more "transformed"  
(cancer-like) they are.

> For proliferation, MTT and DMSO didn't give me a nice standard curve for  
> different cell concentrations.  Could any one please suggest any ideas?

There are many ways to skin that cat:
-Electronic cell counters ("Coulter Counter" after the original  
manufacturer) can give you an estimate of cell number, but can not  
distinguish between life and dead cells. Most labs working on haematology  
have a tool like that.
-FACS can also be used, the scattered light is used to count total cells,  
fluorescein diacetate can be used to specifically label life cells.
-Counting cells under the microscope in a haemocytometer in the presence  
of trypan blue is laborious, but gives very reliable results.
-If life/dead distinction is not important, you may simply spin the cells  
down, wash them in PBS and then determine total protein, DNA or  
phospholipid.


More information about the Methods mailing list