counting and platting of neurospheres - (Oct/30/2007 )
hi you all!
can someone tell me, how is the best and reproducible method to dissociate neurospheres, count the cells and to plate them in 48-well-plates (5000 and 10000 cells/well)? 
I used our cellcounter, but after counting and platting them on the next day, I see, that there are not enough cells each well. I think it is, because the neurospheres are not well dissociate, but I don't know how to do it better (I pipette up and down for 100times and I don't see neurospheres per eye)?! 
I appreciate every advice you can give! Thanx so much!
Hi
is it just mechanical dissociation you do with the pipette? Have you tried using trypsin or even better accutase (less aggressive, doesn't need inactivation). Do you plate the cells on ECM to differentiate them or do you just want to passage them? In the latter case, you should consider the number of cells/ml, which should be around 10000/ml for a clonal dilution.
hope this is of help!
hi!
yeah I just do mechanical dissociation because my supervisor said that enzymes are too aggressive and I should not use them. Yesterday I read a paper about chemical dissociation. have you ever tried this?
I will do an proliferation assay (alamarblue) with the cells, therefore I don't want the cell number too high. I try 10000, 15000, 20000 and 30000 now. So I will see next week if it worked... Maybe you can give me some more advice, that would be very nice 
Thanx a lot!
[quote name='mascat' date='Nov 1 2007, 03:34 AM' post='115699']
Hi
is it just mechanical dissociation you do with the pipette? Have you tried using trypsin or even better accutase (less aggressive, doesn't need inactivation). Do you plate the cells on ECM to differentiate them or do you just want to passage them? In the latter case, you should consider the number of cells/ml, which should be around 10000/ml for a clonal dilution.
hope this is of help!
[/quote
What do you use Accutase on? It is still a protease enzyme.
Hi,
What species are you working on? Some species are easier to dissociate than others. For example, it seems that neurospheres derived from rat cells are much more difficult to dissociate efficiently than those derived from mouse cells, especially at later passages.
We have a method that might help. It is non-mechanical and non-enzymatic. You can download a protocol at: http://www.stemcell.com/technical/28729_MouseDiss.pdf.
Good luck with your research!
Diane Miller
Product Manager, StemCell Technologies