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Cell Splitting Ratio Question - (Jul/25/2012 )

Hello everyone I have a question about cell splitting ratios. I am working at a lab over the summer and I have done cell culture a few times but I am still confused about how to calculate amount of cells to add to my new plates after I collect my cells.
When my C2C12 cells are confluent I remove them from the plate with trypsin and spin it down and keep only the pellet. I add 10ml of new media into a new 10cm plate and 2ml into each well of a 6 well plate. What is the best was to split my cells into ratios like 1:10, 1:15, or 1:20 for each well/plate? What is the best amount of media to add to the pellet to re-suspend and how can I calculate the amount to add to each?

-hungrybipra-

Split ratios don't really work when scalin up or down in flask/plate size. The ratios work like this if you want to do a 1:5 (one part in five) split and you have 10 ml, you take the 10 ml and divide it by 5 to give you 2 ml - transfer 2 ml from the original 10 to a new flask and you have done a 1:5 split.

Of course you can do the same when changing container size, but it becomes a bit meaningless as you are placing into a completely different area. For example taking a 1:2 split from a t-75 flask and placing it into one well of a 6 well plate will result in a completely confluent (actually over-confluent) well, but placing the same into a t-150 flask will only be about 25% confluent...

-bob1-

maybe this is usefull

http://openwetware.org/images/4/47/Cell_Culture_Numbers.pdf

-memari-

hungrybipra on Thu Jul 26 00:49:35 2012 said:


Hello everyone I have a question about cell splitting ratios. I am working at a lab over the summer and I have done cell culture a few times but I am still confused about how to calculate amount of cells to add to my new plates after I collect my cells.
When my C2C12 cells are confluent I remove them from the plate with trypsin and spin it down and keep only the pellet. I add 10ml of new media into a new 10cm plate and 2ml into each well of a 6 well plate. What is the best was to split my cells into ratios like 1:10, 1:15, or 1:20 for each well/plate? What is the best amount of media to add to the pellet to re-suspend and how can I calculate the amount to add to each?


Dear Hungrybipra,

I will expand the advice that memari, in the previous post, has correctly stated.

You should be using cell numbers, rather than a split ratio, to:-

i) Grow your cells

ii) Seeding cells for experiments.

Split ratio's are important in that they give you a rough idea on the "expandibility" of the cells in question.

You are using C2C12 which we also use in our lab. We always split/passage our cells prior to full confluency (as advised by the ATCC information sheet...see link below) :-

http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=crl-1772&Template=cellBiology

Here it gives the correct advice on CELL NUMBERS/SQUARE CENTIMETRE OF GROWING SURFACE i.e. 1.5 x 10(5) - 1.0 x 10(6) cells/ T75cm flask.

The reason why you should use cell number is that you can never accurately estimate how many cells you have at sub confluency/confluency. Again a viable count (Trypan Blue) should be done every time you split/passage your cells as the number of non-viable/dead cells can vary from passage to passage.

Viabilty can be affected by:-

Passage number
Trypsin treatment
CO2 Concentration in the Incubator
Temperature of Incubator
Humidification
FCS batch variation
TC plastic used


Sorry for going on but the way you look after your cells is VITALLY important when setting up and interpreting experimental data.

Hope you find this useful

Kindest regards and good luck.......P.S. Post again if anything is unclear....there are many experts on here (Leeee and bob1) who are more than willing to help cell and tissue culture newbies

Uncle Rhombus

-rhombus-

or this
German cell culture collection
https://www.dsmz.de/...=192

-----
Babak Memari

-memari-

or this

European Collection of Cell Cultures (ECACC)
https://www.hpacultures.org.uk/products/celllines/generalcell/detail.jsp?refId=91031101&collection=ecacc_gc

-----
Babak Memari

-memari-