splitting cells-ideas? - (Mar/16/2005 )

My protocol says :

Hela cells divide once every 24 hours. The split ratio was thus (1/2)^x where x is the number of days to the next cell-culture procedure. When the day comes detach the cells and then add 8ml MEM++ and from the cell suspension, (1/2)^x *8ml was transfered into a new culture flask and MEM++ was added to a total volume of at least 25ml.

My questions:

1. Why is the split ratio (1/2)^x when the cells divide once every 24 hours?

2. What does it mean with (1/2)^x *8ml? Is that the cell number or the volume?

Hope very much for ideas.

Thanks.

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hi
well let say your next cell culture procedure will take place in 3 days.
x=3
The quantity of cells in the plate at 90%confluency is "A".
hence if we assume that there is a division per day, if you seed plate day 1 with A x 1/8 = A/8 [(1/2)^3=1/8]
day 2 there will be 2fold more cells on the plate : A/4 cells on the plate (2 x A/8)
day 3 -> A/2
day 4 (that says 3 days before the previous cell culture procedure) you will get "A" cells on the plate, that leans 90% cinfluency and cells that should be splitted...

that means that the (1/2)^x cells should be in a final volume of 8ml MEM and that you seed the plate with all the 8ml MEM

Fred

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What happens if i use a cell line which divides once every 1,5 days. How would the formula be?




I thought with a split ratio 1/2 means that you make two flasks from one original one. If in this case we take all of the 8ml and cells into another flask then it won't be a 1/2 split ratio, will it?

Thanks for any inputs.

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hi
if cellls divide themselves in 1,5 day, make experiments on them every three days. And the formula would be
(1/2)^(x*3/2)
where 3/2(=1,5) is the fraction representing the PDT

you're right


You pre add 4ml in each new plate or flask
you take your cells in 8ml and put 4ml in each flask!
gently swirl and that will be ok

fred

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Thanks, Fred. But i did not get the correct answer:

(1/2)^(3*1,5)= 0.044. A is 90% confluence.

Day 1-start day: A* 0.044

Day 2: 2*A* 0.044= A*0.088

Day 3: 2* A*0.088=A*0.176

Day 4-working day:2* A*0.176= A*0,352, which is not A.

Any inputs? Thanks.

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hi
i made a little mistake sorry
the formula should be :

(1/2)^(X/PDT), PDT in days.

Hence, should be (1/2)^(X/1,5)=(1/2)^(X/[3/2])=(1/2)^(2X/3)

Your experiment will take place in three days as said in the example.
Seeding : A x (1/2)^(X/PDT) = A x (1/2)^(2X/3) =
with X=3 and PDT = 3/2, and "x" is the multiplication sign

The formula is now :
A x (1/2)^(3 / [3/2])=A x (1/2)^(3 x 2/3)=A x (1/2)^(2)=A x 1/4= A/4

in 1,5 days you'll get : A/2 cells on your plate

in three days you'll get A cells on your plate !


fred

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Thanks.

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What if one of the flasks get more cells than the other. Is that okey?

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hey it's just biology and living cells.
if you perform your try in the afternoon, just look at your falsks in the begining of your lab day and i cells are too concentrate, split them before. but i think that you won't get too diferences between the two flsks
if you want more security, resuspend cells in 16ml, inverse or mix few times too homogeneise. And put 8ml in each flasks. The difference should be less important than reparting 2fold 4ml.


fred

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Thanks again.

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