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Seeding fibroblasts at 1 x 106 in

Seeding density calculation 10-cm dish Fibroblasts

Best Answer bob1, 13 May 2014 - 01:48 AM

Ok - when you first start out it pays to think about what you are trying to find out - if you have 3.02 x 104 cells per ml and 10 ml of that suspension then you have 3.02 x105 cells total (n = c x v, n=3.02x104 cells/ml x 10 ml)...

 

Now as you can see this isn't enough to get 106 cells at all... so you need to grow more cells.

 

However the working for your formula, if you insisted on using it, would be C1=3.02 x 104    V1= ?  C2=106   V2=1 (note you don't want 106 cells/ml and 8 ml of it, you want 106 total).  

V1= (106 x 1)/3.02x104

V1= 33.11 ml

 

V1 is the variable you want to know as it is the amount you need to take from your initial cell suspension to get the concentration and volume you need at the end.   The amount of cells you have initially is irrelevant (unless you don't have enough).  I would usually get people to calculate the total amount of cells the have first (n=c x v) - so that they know the have enough to go on with, then they can calculate further the amount required to get the concentration they need either using C1V1 or v= n/c depending on how they have done the harvest.

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#1 Ahyi

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Posted 12 May 2014 - 06:35 PM

Hi,

 

I'm am seeding fibroblasts at 1x10in 8ml of DMEM in a 10-cm dish.

My initial concentration is 3.02 x 10in 10 ml of resuspended cells. 

I calculated the volume of cell suspension as (3.02 x 104)   x 10 / 1x106 .

Would this be correct?

 

Thanks !

 

 



#2 bob1

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Posted 12 May 2014 - 08:59 PM

If, just like you learned in school, write out the equation you need - then you will make fewer mistakes and be more certain of you results.

 

So... in your case you need two different forms of the equation C=N/V where C=concentration, N=total number (or number you want) and V=volume.

 

Can you work it from there?

 

Check out the many many other threads on dilutions and cell counting on here too.



#3 Ahyi

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Posted 13 May 2014 - 12:20 AM

Thanks Bob1 - for the prompt reply.

I worked with the formula C1V1= C2V2 

Where C1 = 3.02 x 104 , V1=? 

            C2 = 1x106   , V2 = 10

 

This gives me 331.12 ml - too large a volume for my 10 cm dish. I need to know how much of the cell suspension (containing 3.02 x 104

 cells / ml ) that I will need to make up to 8 ml with medium.

There's something I am missing. Been through several posts here and elsewhere. It gets more confusing. 

If i could see the actual calculation with an explanation - I'd be mighty glad and less likely to pull my hair out!



#4 bob1

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Posted 13 May 2014 - 01:48 AM   Best Answer

Ok - when you first start out it pays to think about what you are trying to find out - if you have 3.02 x 104 cells per ml and 10 ml of that suspension then you have 3.02 x105 cells total (n = c x v, n=3.02x104 cells/ml x 10 ml)...

 

Now as you can see this isn't enough to get 106 cells at all... so you need to grow more cells.

 

However the working for your formula, if you insisted on using it, would be C1=3.02 x 104    V1= ?  C2=106   V2=1 (note you don't want 106 cells/ml and 8 ml of it, you want 106 total).  

V1= (106 x 1)/3.02x104

V1= 33.11 ml

 

V1 is the variable you want to know as it is the amount you need to take from your initial cell suspension to get the concentration and volume you need at the end.   The amount of cells you have initially is irrelevant (unless you don't have enough).  I would usually get people to calculate the total amount of cells the have first (n=c x v) - so that they know the have enough to go on with, then they can calculate further the amount required to get the concentration they need either using C1V1 or v= n/c depending on how they have done the harvest.



#5 Ahyi

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Posted 13 May 2014 - 05:41 PM

Thanks bob1 - again.

Now I get it - clearly.

 

Wow, glad I joined this forum.



#6 mlomonaco

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Posted 09 July 2014 - 03:45 PM

Ahyi...take a deep breath and chill! Each investigator must empirically determine cell culture conditions.Here is a back of the envelope calculation: 3E5 cells in 10mls will double 5 times
in 5 days to yield 10 million cells. Now make us proud!





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