7 replies to this topic

[sagagirish]

Hi....i have been growing HeLa cells ....they look fine...but when i trypsinize them,,,most of them detach to form single cells..however some huge clumps of cells are formed ...and this causes loss of huge amount of cells..how is it that there are no clumps initially and later clumps are formed?
Is this bad ?

Thanks

[Inmost sun]

sagagirish, on Jun 21 2010, 05:42 PM, said:

Hi....i have been growing HeLa cells ....they look fine...but when i trypsinize them,,,most of them detach to form single cells..however some huge clumps of cells are formed ...and this causes loss of huge amount of cells..how is it that there are no clumps initially and later clumps are formed?
Is this bad ?

Thanks

do you wash cells with serum-supplemented cell medium to inactivate trypsin? normally, albumine reduces clumping...

[leelee]

Do you pellet your cells and remove the trypsin? You should be able to seperate your clumps by pipetting up and down several times when resuspending the cell pellet after washing off the trypsin.

[sagagirish]

Inmost sun, on Jun 21 2010, 12:29 PM, said:

sagagirish, on Jun 21 2010, 05:42 PM, said:

Hi....i have been growing HeLa cells ....they look fine...but when i trypsinize them,,,most of them detach to form single cells..however some huge clumps of cells are formed ...and this causes loss of huge amount of cells..how is it that there are no clumps initially and later clumps are formed?
Is this bad ?

Thanks

do you wash cells with serum-supplemented cell medium to inactivate trypsin? normally, albumine reduces clumping...

Actually ..yes i do inactivate the trypsin with a complete medium..i can atually start seeing hude clumps of cells with my naked eyes...

[sagagirish]

leelee, on Jun 21 2010, 11:14 PM, said:

Do you pellet your cells and remove the trypsin? You should be able to seperate your clumps by pipetting up and down several times when resuspending the cell pellet after washing off the trypsin.

yaaa....after adding trypisn..i keep my flask in incubator and check every 2 mins and keep keep the trypsinisation for abt 10 mins before i stop it with fresh medium ...by that time ,,,too many clumps...when i observe under microscope ,its a huge mass or strand of cells that aggregated .

I do resuspended them after centrifugation but it tough to break them after they formed clumps...so basically i lose a lot of cells in these clumps...
am i doing soemthing wrong somewhere?

[bob1]

sagagirish, on Jun 22 2010, 10:06 AM, said:

yaaa....after adding trypisn..i keep my flask in incubator and check every 2 mins and keep keep the trypsinisation for abt 10 mins before i stop it with fresh medium ...by that time ,,,too many clumps...when i observe under microscope ,its a huge mass or strand of cells that aggregated .

I do resuspended them after centrifugation but it tough to break them after they formed clumps...so basically i lose a lot of cells in these clumps...
am i doing soemthing wrong somewhere?

It sounds like you are trypsinising for too long, you don't need 100% of the cells to be detached before you can start adding the medium to neutralise, as the washing action from squirting the medium in should mechanically remove most of the still attached ones.  Try adding trypsin and giving the flask a few taps on the side when you see the first cells lifting off.

[leelee]

I agree that your trypsinisation is way too long- I usually do around 1 or 2 min max. I wonder if you are killing a portion of your cells?

Do you tap your flask to dislodge the cells when you are checking them during trypsinisation? I usually just wait until the cells are rounded up (but still attached) then give the flask a good sideways tap on the heel of my palm and then check again.

Maybe you could try adding some EDTA to your trypsin to help speed it up?

[sagagirish]

leelee, on Jun 22 2010, 07:55 PM, said:

I agree that your trypsinisation is way too long- I usually do around 1 or 2 min max. I wonder if you are killing a portion of your cells?

Do you tap your flask to dislodge the cells when you are checking them during trypsinisation? I usually just wait until the cells are rounded up (but still attached) then give the flask a good sideways tap on the heel of my palm and then check again.

Maybe you could try adding some EDTA to your trypsin to help speed it up?

thanks a lot guys...i had read that i should keep trypsinisation for around 10 mins...and that why i was doing it...so maybe this time i will do it for 2 mins...my trypsin is actaully a 1X trypsin-EDTA mix...