Getting Cells to adhere STRONGLY - help for high throughput screen (Mar/20/2008 )
Hello-
We are going to set up a high throughput screen, but are having problems keeping our cells attached to the plate. We are using HEK293 Griptite cells. Griptite cells are just commercially available HEK293's transfected with the macrophage scavenger receptor, which increases adherence. This, however, is not enough, as or liquid handling robot keeps blowing off the cells unless they are at very high confluency.
Please, Does anyone know a trick to further increase the adherence of our cells???? We have tried polydlysine already, and are going to try fibronectin and a product called cell-tak in the next few weeks. We have already optimized the robot and everything, so we unfortunately cannot make it any more gentle.
In addition to commercially available coatings, we were thinking of coating with some sort of ligand for the macrophage scavenger receptor (Oexpressed in griptite cells), but unfortunately most of them are things that are toxic or dont want to add to our cells routinely for our screen.
Does anyone have any good ideas for getting these cells to stick? Simple or otherwise?
Thanks very much,
Mountainman
We are going to set up a high throughput screen, but are having problems keeping our cells attached to the plate. We are using HEK293 Griptite cells. Griptite cells are just commercially available HEK293's transfected with the macrophage scavenger receptor, which increases adherence. This, however, is not enough, as or liquid handling robot keeps blowing off the cells unless they are at very high confluency.
Please, Does anyone know a trick to further increase the adherence of our cells???? We have tried polydlysine already, and are going to try fibronectin and a product called cell-tak in the next few weeks. We have already optimized the robot and everything, so we unfortunately cannot make it any more gentle.
In addition to commercially available coatings, we were thinking of coating with some sort of ligand for the macrophage scavenger receptor (Oexpressed in griptite cells), but unfortunately most of them are things that are toxic or dont want to add to our cells routinely for our screen.
Does anyone have any good ideas for getting these cells to stick? Simple or otherwise?
Thanks very much,
Mountainman
It will be hard to achieve. 293 cells are easy to come off even if you fix them chemically. Maybe you have to think about to switch to another cell line that attachs better.
The Griptite version is much better than normal HEK293s. So I'm hopeful we can tweak it with a coating or something. Making a new cell line would be a last resort.
Any other (more attractive) ideas?
Any other (more attractive) ideas?
cell-tak will fix the cells to a distinct position; motility will not be able (if this is interetsing to you); unspecific stimulations by cell-tak fixation may be possible;
3D cell culture in Matrigel or collagen may be an alternative
Any other (more attractive) ideas?
cell-tak will fix the cells to a distinct position; motility will not be able (if this is interetsing to you); unspecific stimulations by cell-tak fixation may be possible;
3D cell culture in Matrigel or collagen may be an alternative
fixing them in place would be ok....we would be doing a high content immunflourescent screen (which requires <90% confluent monolayer) so we couldn't go 3D
Can you bend the needle or the tip such that the liquid does not go straight towards the cells, rather to the wall? I have never worked with a robot before, just a wild guess.
Interestingly for this cell line fibronectin seems to work acceptably well. The best adherence coatings for these cells were
Fibronectin> FBS>Collagen>Poly-D-Lysine
Interesting that poly-d-lysine works the least well....
Fibronectin> FBS>Collagen>Poly-D-Lysine
Interesting that poly-d-lysine works the least well....
beware that fibronectin activates integrins and, therefore, integrin-dependent processes
Not terribly familiar with ECM biology. Integrins can signal? 
Would you be willing to throw a reference my way for someone who is uninformed? Maybe we will use FBS then...I will consult my colleagues...
Would you be willing to throw a reference my way for someone who is uninformed? Maybe we will use FBS then...I will consult my colleagues...try these reviews:
JCS review
Curr Op Cell Biol 2006