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# Passage ratio 1:3

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### #1 cupidstunt

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Posted 08 May 2009 - 08:38 AM

Hi,

I have a very basic question. I am starting to do cell cultures and I have come across different ratios. What does 1:3 ratio mean? How to passage you cells in 1:3 ratio or say 1:4 ratio in 25 ml of media?

Thanks

### #2 kbee

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Posted 08 May 2009 - 09:49 AM

cupidstunt, on May 8 2009, 09:38 AM, said:

Hi,

I have a very basic question. I am starting to do cell cultures and I have come across different ratios. What does 1:3 ratio mean? How to passage you cells in 1:3 ratio or say 1:4 ratio in 25 ml of media?

Thanks

The ratio just refers to the ratio of confluent cells to fresh media used when passaging/splitting the cells. A 1:3 ratio indicates 1 part confluent cell culture to 3 parts fresh media.  In a 25mL culture, this would be ~6mL of the original cell culture and ~18mL of fresh media.  THe ratio you choose will depend on what seeding density your cells prefer, how quickly they divide and how long you want to leave them until the next split.

### #3 cupidstunt

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Posted 11 May 2009 - 08:33 AM

thanks!

Edited by cupidstunt, 11 May 2009 - 08:34 AM.

### #4 rhombus

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Posted 12 May 2009 - 01:03 AM

cupidstunt, on May 8 2009, 05:38 PM, said:

Hi,

I have a very basic question. I am starting to do cell cultures and I have come across different ratios. What does 1:3 ratio mean? How to passage you cells in 1:3 ratio or say 1:4 ratio in 25 ml of media?

Thanks

A split Ratio of 1:3 for cell culture means:

1 flask of cells 80% confluent will need to be split into 3 flasks at 25/30% confluent.
If you exceed this ratio i.e. do a 1:6 split, then the cells may not recover because the number of cells/flask is too low.

IT HAS NOTHING TO DO WITH THE VOLUME OF MEDIA!!!!!!!!!!!

The volume of media is important when the cells are first split. In a T25cm, the volume should be no more than 5mls. As the cells grow and become more confluent, the volume of media should rise to a maximum of 12mls.

the "bible of cell culture " (Freshney) states:

0.2-0.5ml/square cm of growing surface

Regards

Rhombus

### #5 leelee

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Posted 12 May 2009 - 08:38 PM

I second Rhombus' post. (and I'm happy to see you are still here, hadn't seen a post from you in a while and was getting worried!!)

### #6 cupidstunt

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Posted 13 May 2009 - 06:41 AM

rhombus, on May 12 2009, 10:03 AM, said:

cupidstunt, on May 8 2009, 05:38 PM, said:

Hi,

I have a very basic question. I am starting to do cell cultures and I have come across different ratios. What does 1:3 ratio mean? How to passage you cells in 1:3 ratio or say 1:4 ratio in 25 ml of media?

Thanks

A split Ratio of 1:3 for cell culture means:

1 flask of cells 80% confluent will need to be split into 3 flasks at 25/30% confluent.
If you exceed this ratio i.e. do a 1:6 split, then the cells may not recover because the number of cells/flask is too low.

IT HAS NOTHING TO DO WITH THE VOLUME OF MEDIA!!!!!!!!!!!

The volume of media is important when the cells are first split. In a T25cm, the volume should be no more than 5mls. As the cells grow and become more confluent, the volume of media should rise to a maximum of 12mls.

the "bible of cell culture " (Freshney) states:

0.2-0.5ml/square cm of growing surface

Regards

Rhombus

hi!