best con. of trypsin with neuro cells? - (Nov/12/2007 )
what is the best concentraion to used trypsin-EDTA with neuro cells?
i tired 0.25 % it was too harsh ...theni tried 0.025 % still it is 
...shall i try lower concentraion ? !!
thanx
Muna
i have used 0.05% and 0.25% for neuronal cells. My cells seem to tolerate both of the conc. fine.
how did you conclude that the cells are not doing fine.
yes 0.05% is normal, just try decrease time to expose the cells on tripsin
Siva
sorry fot not replying .....
i come to know that the cells are not fine because....after trypsinization ...i get a lot of did cells
and there will be in the next morning a cellular debris where the media is clear and red color and
after a week or two it start not to adhere after trypsinization.
the trypsin-EDTA i used 0.05 % which is too harsh on cells and clumps formation.
0.005 % i got clumps .
0.0005 % i got single cells but alot of dead cells .
i used 1.5 ml for T75 flask..for 2 min
wht shall i do....dilute it more ?
i come to know that the cells are not fine because....after trypsinization ...i get a lot of did cells
and there will be in the next morning a cellular debris where the media is clear and red color and
after a week or two it start not to adhere after trypsinization.
the trypsin-EDTA i used 0.05 % which is too harsh on cells and clumps formation.
0.005 % i got clumps .
0.0005 % i got single cells but alot of dead cells .
i used 1.5 ml for T75 flask..for 2 min
wht shall i do....dilute it more ?
I am guessing some other solution is the cause for this problem. Are you sure about the media which you are using for the cells. Is it the right media for these cells.
Does your media have FBS?
yes ...i added 10% FBS + 5 % horse serum..
Have the serum been tested for performance ? An untested batch could cause problems too.
You should try to tritrate cells for longer. This helps to prevent clump formation.
yes serum is tested ...
indeed iam using 0.0005% trypsin-edta for 5-6 min it is much ok.
...i have a Q
how many passages i go further....i mean at what passage i should stop with neuro cells?
yes serum is tested ...
indeed iam using 0.0005% trypsin-edta for 5-6 min it is much ok.
...i have a Q
how many passages i go further....i mean at what passage i should stop with neuro cells?
Each cell line is different. usually when people start to notice difference in the cell like growth rate, different behavior, morphology etc., they stop culturing them. Also it depends on how they are cultured through out which can determine the longevity of the cells.