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Cell counting issues


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#1 autoclave239

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Posted 27 August 2009 - 05:21 AM

Hi all,
I'm sure other people have this problem. I work with CHO cells and use those plastic Corning Erlenmeyer flasks, and when I do exhaustions (where I let the flask shake with cells for several days without splitting) I count my cells every day. Problem is, as the days go on (usually within the first two days) a "ring of death" forms around middle of the flask where the liquid sloshes against the wall. When I count, I mix thoroughly with a 10 ml pipette, but the angle of the flask wall means that I can only get a weak stream of liquid to splash against the dead ring part. This really messes up my counting as the dead cell count is just as important as the live one. Does anyone know how to get these cells off the flask so I'm able to count them?

#2 bob1

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Posted 27 August 2009 - 04:18 PM

Use trypsin to detach, or use a cell scraper?

#3 autoclave239

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Posted 28 August 2009 - 04:06 AM

Use trypsin to detach, or use a cell scraper?


I've thought of both, but trypsin will probably skew the exhaustion results (i.e. kill more cells than would die naturally). Cell scrapers are going to take the cells off, but I was told by my advisor if I do that I will end up shearing and ultimately destroying those cells, which means I can't count them. I guess I'm looking for a technique that people use to get a good hard stream of liquid against the side of the flask (I kind of wish I had an "L" shaped pippette tip, but I don't think such a thing exists)

#4 Carlton H

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Posted 31 August 2009 - 04:42 PM

If the "ring of death" is stuck to the flask, can you just take a small (negligible?) volume of media out of the flask and then count the cells in that volume?

-Carlton

Edited by Carlton H, 31 August 2009 - 04:45 PM.

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#5 autoclave239

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Posted 01 September 2009 - 05:41 AM

If the "ring of death" is stuck to the flask, can you just take a small (negligible?) volume of media out of the flask and then count the cells in that volume?

-Carlton


Actually, that's exactly what I do. But the cells from the dead ring are still not included in this small/negligible volume. This wouldn't be a big deal if the ring didn't get so thick with dead cells that need to be included in my calculations.

#6 rhombus

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Posted 02 September 2009 - 04:16 AM

If the "ring of death" is stuck to the flask, can you just take a small (negligible?) volume of media out of the flask and then count the cells in that volume?

-Carlton


Actually, that's exactly what I do. But the cells from the dead ring are still not included in this small/negligible volume. This wouldn't be a big deal if the ring didn't get so thick with dead cells that need to be included in my calculations.



This is a bit left field but can you Siliconise your Erlenmeyers? We do this with our Techne glass stirrers.....to stop the cells attaching to the sides of the bottle.

You could do that with Glass Erlenmeyers and then autoclave them..

Reusable...therefore saving money on disposable flasks
Easy to do and takes little time.

Regards

Rhombus




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