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Passaging Cells


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#1 labrat612

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Posted 14 January 2010 - 12:11 PM

So I have a question for you guys out there...

I've been doing the same splitting procedure for my CHO and 293 cells for years. Normally, after the cells have detached due to the addition of trypsin, I add serum-containing media to neutralize the trypsin. Then, I spin the suspension at 1000 rpm for 5 mins at 4c.
Now, I had learned to do it at 4C because it reduces some enzymatic and proteolytic activity. However, I am beginning to wonder if that is actually true. And perhaps spinning them at 4C is more of a shock to their system because its so cold vs doing it at room temp.
Or does it even matter-- after all, it's only for 5 minutes.

What are your thoughts?


~Labrat612

#2 bob1

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Posted 14 January 2010 - 03:16 PM

I only spin my cells at 4 deg C if I am harvesting them for use in an experiment and need to stop/slow metabolism.

Short exposure to cold will induce upregulation of cold-shock genes which protect the cell from damage. I suspect that this will be happening with your cells, but the effects should go away after 12 hours or so (I think).

#3 goldfinger

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Posted 19 February 2010 - 12:23 PM

I do not spin them down. I just add a small fraction to new media (usually 1:10)

#4 scolix

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Posted 19 February 2010 - 11:43 PM

After trypsinisation, I add media with serum to neutralise it and split cells accordingly. I never spin them down. Its worked for me all the time. Why do you need to spin them down?
Saves time and my cells are fine.

#5 Dukey

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Posted 22 February 2010 - 03:25 PM

I always spin down at RT for splitting, never at 4 degrees. It seems pretty much the standard way that most labs do it.




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