4 replies to this topic

[9939]

Hi all,

I'm currently working on Lu165 cell line (suspension cells). However, the cells form huge clumps which is very difficult to break even if I pipette up and down vigorously for about 10 times. This brings to the question of difficulty in cell counting and subculturing as single cell suspension is hard to achieve. And I noticed, those plates with lots of cell clumps enters quiescent soon and the cells die off after that.     

Do I add any chemicals to break the clumps? If so, what's the concentration and for how long?

Also another silly question - how to remove cell debris efficiently with suspension culture? I tried to centrifuge and aspirate the media but I don't think that works really well. The cell debris gets more and more as the passage number increase. I could be wrong - but will that affect the cells? It gets pretty scaring when you thought the cells is contaminated when you mistaken the cell debris as some microbes! LOL.

Any suggestions would be useful. Thanks~

[Dr Teeth]

wia84, on Dec 11 2009, 07:37 PM, said:

Hi all,

I'm currently working on Lu165 cell line (suspension cells). However, the cells form huge clumps which is very difficult to break even if I pipette up and down vigorously for about 10 times. This brings to the question of difficulty in cell counting and subculturing as single cell suspension is hard to achieve. And I noticed, those plates with lots of cell clumps enters quiescent soon and the cells die off after that.     

Do I add any chemicals to break the clumps? If so, what's the concentration and for how long?

Also another silly question - how to remove cell debris efficiently with suspension culture? I tried to centrifuge and aspirate the media but I don't think that works really well. The cell debris gets more and more as the passage number increase. I could be wrong - but will that affect the cells? It gets pretty scaring when you thought the cells is contaminated when you mistaken the cell debris as some microbes! LOL.

Any suggestions would be useful. Thanks~

Try adding EDTA to a final concentration of 5 mM, before triturating your cells.  You can them run your cell suspension through a 40 uM nylon cell strainer (depending on your cell size).  Falcon makes some (REF 352340) that fit on top of a 50 ml tube and will not allow cell clumps to pass.

[rhombus]

Dr Teeth, on Dec 14 2009, 07:38 PM, said:

wia84, on Dec 11 2009, 07:37 PM, said:

Hi all,

I'm currently working on Lu165 cell line (suspension cells). However, the cells form huge clumps which is very difficult to break even if I pipette up and down vigorously for about 10 times. This brings to the question of difficulty in cell counting and subculturing as single cell suspension is hard to achieve. And I noticed, those plates with lots of cell clumps enters quiescent soon and the cells die off after that.     

Do I add any chemicals to break the clumps? If so, what's the concentration and for how long?

Also another silly question - how to remove cell debris efficiently with suspension culture? I tried to centrifuge and aspirate the media but I don't think that works really well. The cell debris gets more and more as the passage number increase. I could be wrong - but will that affect the cells? It gets pretty scaring when you thought the cells is contaminated when you mistaken the cell debris as some microbes! LOL.

Any suggestions would be useful. Thanks~

Try adding EDTA to a final concentration of 5 mM, before triturating your cells.  You can them run your cell suspension through a 40 uM nylon cell strainer (depending on your cell size).  Falcon makes some (REF 352340) that fit on top of a 50 ml tube and will not allow cell clumps to pass.

Hi Dr. Teeth....


I do love that word "TRITURATION".....

I have always remove cell debris by just allowing the cells to sediment in a centrifuge tube ....and then removing the supernatant.  You will of course lose a small percentage of the cells. However centrifugation will only sediment the cells AND DEBRIS.

Hope this is useful

Kindest regards

Rhombus

[ChrisHarris]

rhombus, on Dec 15 2009, 03:23 AM, said:

Hi Dr. Teeth....


I do love that word "TRITURATION".....

I have always remove cell debris by just allowing the cells to sediment in a centrifuge tube ....and then removing the supernatant.  You will of course lose a small percentage of the cells. However centrifugation will only sediment the cells AND DEBRIS.

Hope this is useful

Kindest regards

Rhombus

What kind of times/volumes are you typically dealing with to allow efficient sedimentation?
   Cheers
Chris

[rhombus]

ChrisHarris, on Dec 16 2009, 10:57 AM, said:

rhombus, on Dec 15 2009, 03:23 AM, said:

Hi Dr. Teeth....


I do love that word "TRITURATION".....

I have always remove cell debris by just allowing the cells to sediment in a centrifuge tube ....and then removing the supernatant.  You will of course lose a small percentage of the cells. However centrifugation will only sediment the cells AND DEBRIS.

Hope this is useful

Kindest regards

Rhombus

What kind of times/volumes are you typically dealing with to allow efficient sedimentation?


Within 5 minutes most of the live cells will sediment....disrupted cells/cell debris will still be floating in the supernatant.

Regards

Rhombus

   Cheers
Chris