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# Calcutation question

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### #1 yyc

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Posted 08 October 2013 - 11:09 AM

Hi,

I am a first year PhD student.

I have a calculation question:

I have to culture cells in 1 ml and then I have to add some stimulators and enhancers. I have 3 of these stimulators and the final concentration in the cell culture for each stimulator should be: Stimulator A: 1.2 mg/ml; stimulator B: 1 mg/ml; stimulator C: 2 mg/ml.

If I make the calcultation for stimulator A (stock concentration is 120 mg/ml), then I have to add 10.16 microlitres, following the equation V1 x C1 = (V2 + V1) x C2:

V1 x 120 mg/ml = (1ml + V1) x 1.2 mg/ml

But now I have to add stimulator B (stock concentration 100 mg/ml) and I have a volume of 1011.6 microlitres in the cell culture due to the volume added with stimulator A. Doing the same equation:

V1 x 100 mg/ml = (1.011 ml + V1) x 1 mg/ml

That makes 10.2 microlitres of stimulator B that I have to add.

But the problem now is that I have a increase of 10.2 microlitres in the volume of my cell culture due to stimulator B,  therefore stimulator A is not at 1.2 mg/ml anymore due to the increase of volume with stimulator B. And then the same problem when I add stimulator C.

Can anybody help me with these calculations?

thanks. YYC

No need to triple post things - I've deleted your other two identical posts!  Bob.

### #2 jerryshelly1

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Posted 08 October 2013 - 12:27 PM

You just need to pick a total final volume and make all other calculations based on that. You want your total volume to be 1mL of media? Make all of your calculations based on 1mL of media and subtract the amount of stimulator A, B and C from your 1mL of media.

Do you need to add your stimulators at different time points or are they all added at the exact same time.  If they are added at different time points, the 10.2uL difference will be negligible as the amount of stimulator A will have decreased due to your cells consuming it.

Does this make sense?

### #3 DRT

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Posted 08 October 2013 - 04:37 PM

It is not going to make a practical difference but to be precise:

Each of your 3 stimulators is going to be diluted by 1/100

This means the 1mL cell volume will be 97/100 of the final volume (1mL/.97 = 1.031mL)

Add 10.3µL of each of the stimulators (31µl/3)

I’m not sure how you got 10.16µL for a single stimulator it should be 10.10µL

### #4 yyc

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Posted 13 October 2013 - 10:06 AM

Thank you jerryshelly1 and DRT for you explanations and help.

DRT, I am quite interested in your calculations of the final volume: What happen then if my stimulators have to be diluted by different factor. For example: stimulator A 1/20; stimulator B 1/100; stimulator C 1/200.

For 1 ml of cell culture. How I calculate the final volume?

Sorry, maybe this is quite simple question, but it is something you don't dare to ask and then you acumulate this doubt for long time.

Thank you!

### #5 DRT

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Posted 13 October 2013 - 11:36 AM

If you are doing the calculations on the fly at your bench and with a basic calculator the simplest way is to switch all the dilutions to a common denominator.

In this case:

The 1mL of cell culture would be 187/200 of the final volume.

Which makes the final volume = 1000µL x (200/187) = 1069.5µL

Each 200th of 1070 is 5.35    so A=53.5µL B=10.7µL C=5.3µL

The key is not to try and remember the calculations but to picture the whole process so that you already have a rough idea of what the answer is going to be before hitting the calculator.

Good luck,

### #6 yyc

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Posted 14 October 2013 - 09:36 AM

Got it!

Thank you very much DRT. Very useful.