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Trypsinizing and Subculturing Mammalian Cells

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Cite this Article: Trypsinizing and Subculturing Mammalian Cells

Ricardo, R., Phelan, K. Trypsinizing and Subculturing Mammalian Cells. J. Vis. Exp. (16), e755, doi:10.3791/755 (2008).

Abstract: Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

Protocol: Trypsinizing and Subculturing Mammalian Cells

The complete text protocol for this experimental approach is available in Current Protocols in Cell Biology.

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Disclosures: Trypsinizing and Subculturing Mammalian Cells

The authors have nothing to disclose.

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11 Comments

the video isnot playing

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Posted by: AnonymousJuly 6, 2008, 10:23 PM

how to prepare cell growth curve of  cancer cell line

2

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Posted by: amitSeptember 11, 2008, 3:10 AM

can i know the passaging protocol for MDA MB 231 Breast cancer cells.

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Posted by: navSeptember 18, 2008, 12:10 PM

sir can u tell me that how to transfer cell ( frozen into liq. nitrogen) into media and that will be the mother culture so in that also we have to do passaging , means we have to add HBSS and Trypsin and EDTA. and how to count cells.

thank you

waiting for your reply....

sam A Masih

5

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Posted by: samSeptember 24, 2008, 12:24 PM

Hi,

For subculturing suspension cells, what is the normal routine? is it better if changed everyday or only after the media contents turns trubid and yellowish colour?

Regards

Kamal

6

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Posted by: kamal prajapatiOctober 22, 2008, 7:45 PM

Hello kamal bro..
paras sir le bhannu bhayeko ta tyahi ho kya re...
hemanta

6.1

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Posted by: hemantaJuly 28, 2009, 7:18 AM

Thank you

7

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Posted by: AmmarMarch 24, 2009, 4:09 PM

Could you elaborate on how to culture cancer cells? The precautions to be taken and when to subculture them.

Thank you

8

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Posted by: Preethi S.April 2, 2009, 12:37 PM

I can see the video

9

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Posted by: RFJune 22, 2009, 12:25 PM

We were having a technical issue when you posted your comment. Please let us know at support@jove.com if you are unable to view this video.

9.1

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Posted by: AnonymousJune 23, 2009, 7:52 PM

every time I trypsinize my BBe cells - they clump together (even I don't shake the flask at all..)
how can I prevent it ? because the clumps are super strong and I can not brake them down again.. I do something wrong?
I wash 2x with PBS (37C) and 5-15min trypsinize the cells and add RPMI 1640 media carefully to it and mix gently..

can you help me?

thank you
AlexK

10

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Posted by: Alexandra K.April 12, 2010, 5:19 PM

thank u

11

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Posted by: mohamed ragabOctober 21, 2010, 6:32 AM

How long does it take for an MCF-7 cell lines grow confluent? What's the best media for culturing this cell?
Thanks!

12

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Posted by: TinaSeptember 22, 2011, 11:44 AM

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