Seeding plates - (May/06/2011 )

Hello,

Hope you can help me in seeding my culture plates.
The problem is with how much cells i need to seed the culture plate

Number of cells is 24*10000 cells/ml when counted and In 12 well plate i need to seed 6250000 cells in a total volume of 2500ul per well.
I am confused here of how to carry on with this part.

Please help me.

I have seen a post here with the same problem as mine but i could not understand it.

There are lots and lots of threads on this site about how to calculate cell dilutions...

The answer is to know formulae... C1V1= C2V2 where C1 =initial concentraction, V1= initial volume and C2 and V2 are the concentration and volume you want.

Note that you didn't supply a volume of the cells you have - which you need to be able to calculate the dilution. If you only have one ml of cells, you do not have enough cells to do your dilution.

I am sorry for that.
I resuspended the cells in 20ml and i finally want to plate in each well abt 6250000 cells and like this i want to plate 12 wells.

But wen i go for calculation as you said....

C1V1=C2V2
240000*V1=250000(FOR 1 ML)* 30 ML(for 12 wells,each well of 2.5ml)
V1=30*250000/240000
V1=30*25/24
V1=31.25ML

But how can i take 31.25ml from 20ml stock solution???

Hope you understood where my problem is and hope you correct me where i went wrong.

kumara1984 on Sat May 7 08:25:57 2011 said:

Their is a post similar to this one.. check what I and other posted there....

You cant do this the way you are calculating this.

Have you read every message in that post? Because if you did, you must for sure now understand what the problem is when you see that you need 31ml and only have 20ml .....
Spin sample down and.... check the other topic.
(what is it that you dont understand? because if you dont tell us, we might reply the same way as in that other topic, and it wont help you at all then)

I get

C1= 240000, V1= ?

C2= 250000 V2= 12 (as you said you want to plate 250000/well, you can dilute this to 2.5 ml/well later or you can calculate the concentration and substitute that in for C using V=N/C, where V and C as before and N is the number of the things you want... these are the same formulae you use for chemical dilutions too).

Therefore

V1= 250000 *12/ 240000

V1= 12.5 ml

So if you want to dilute this out to 2.5 ml/well x12 wells = 30 ml, take 17.5 ml of medium and add 12.5 ml of your initial number of cells. Remember to mix the cells before taking out aliquots, the settle very fast.

kumara1984 on Sat May 7 08:25:57 2011 said:

hi kumara....welcome to bioforum....and you're not the only math atheist here (I had to delete my previous reply here bec I understood wrong therefore calculated wrongly)

based on your count, you only have a total of 5 million cells (250,000 cells X 20 ml) so you don't even have enough to put in one well (even if you spin them down again)....

- you can do a re-count of your cells (just to make sure that you didn't make a mistake in counting)
- expand your cells more so you'd have a higher concentration of stock to start with....or resuspend them in less volume...
- or you could probably seed less...it depends of course on the kind of cells you have and what you plan to do with them...

bob1 on Sat May 7 23:29:32 2011 said:

hmmmm....this is getting more exciting.....hi bob..if you go back to his/her first post...it's 6.25 million in 2.5 ml so I think it's a typo...it's 2.5 million/ml the final concentration and not 250,000....and then it will still have to be 30 ml the final volume, I think....he/she still doesn't have enough cells in the end...

Just ignoring the maths for a moment- Am I reading this right? Do you really want to seed 6250000 cells into each well of a 12 well tray?
So 6 million cells per well? Crazy talk!! A confluent well of a 12 well tray/plate will only hold about 1/2 million cells.

Yeah, that's what I was thinking too, which is why I thought in the second post that they were saying 250000/well and that the 6250000 was a typo...

Not to mention that 2.5 ml is too much for a well of a 12 well plate, it's too much even for a well of a 6 well plate where 1-2 ml is perfect.

bob1 on Sun May 8 07:55:01 2011 said:

If you check the other topic then they mean 625.000 cells per well .....
I wondering what it has to be now...

Do they mean 6.250.000 cells/ well or 625.000 cells/Well
or do they mean 625.000 cells/plate (thus for 12wells)


Either they didnt understand the question for the lab excercise or its just a theoretical calculation ...

bob1 on Sat May 7 23:29:32 2011 said:

I agree on the calculation, but its even more confusing.
And I dislike the use of formulae if people dont understand them and I dont think the topicstarter undesrtands it.

I'll break up the formulae for that.


Lets look at the fact , the things we know:

You know that you have 240.000 cells/ml and that in total you have 20 ml. And we know we have a 12 well plate and that every well holds 2,5ml

What is asked? = you need to fill a 12 well plate with 6.250.000 cells/well.

Ok, how do we solve it?

6.250.000 cells per well and 12 wells equals 75.000.000 cells in total.
So we need to have 75.000.000 cells in total, this means for a total volume of 30 ml, since 12wells*2,5ml/well is 30ml.
75.000.000 cells/30ml means= 2.500.000cells/ml
Now if you take back the original concentration of 240.000 cells/ml you see directly its impossible to go from a lower concentration to that higher concentration!
(and when you are in doubt , check it with absolute numbers: check how many cells you have at the start : 20ml*240.000 cell/ml = 4.800.000 cells , this is by far not the 75.000.000 cells you need!!!)

Now lets assume you made a type and you mean 250.000cells/well
(if its not correct, it doesnt matter, you just take another number then and to the calculation again, but you know how to now)

Then we start again:

250.000 cells/well equals for 12 wells: 3.000.000 cells, so I need this amount of cells and my stock is 240.000 cell/ml and I have 20ml.

the question now is: how much ml do I need to take from this stock to have 3.000.000 cells? easy: its
12,5 ml...3.000.000cells/(240.000cell/ml)= 12,5ml
Now I know that the end volume has to be 30ml... so I just add water or medium till I reach 30ml.


So you see: I never "used" the C*V=C*V formulae (I did, but I did it by reasoning).

Dont think about formulae, try to understand it.... this is how you solve questions!
And write down what each number is! Write the units down! You cant make mistakes writing down units!

To explain why the units are important, you need to look at the formula that bob1 used.

The general formula is: C1*V1 = C2*V2

And this means: concentration (cells/ml) * volume (ml).

Now on the right it is still ok

(C1= 240000, V1= ? )

But on the left he changes the units from Cell/ml to cells/well! So the V is no longer a volume but a "number" (wells), thus its 12 from 12 well.

(C2= 250000 V2= 12 and here C : cells/well and V = #wells)

And he uses the formula: N=C/V (cells= cells/ml /ml) but even if you dont "know" this formula, its very simple to get to. Its simple logic.
And the reason he uses it is because he things that the given is not 250.000 cells/ml but 250.000 cells/well... so he doesnt know the "concentration" (in volume) and because he doesnt know it, he uses this given.
But in order to use this in the C*V=C*V formula , he needs to adjust the V too.... (he knows this by looking at the units!!!)


Anyway: I "NEVER" use C*V=C*V , I never write this formula down... why? Because you can make mistakes using formulaes while not thinking.

I always look at the given fact , write down the units and then I think about what I need/want...

Lets look at another example (and why we use this formula).

I need 5moles/ml and 3 ml of this to do my test.
And I know that in the cabinet I have a stock solution of 20 moles/ml.
So what do I do?
Yeah, most would say: C1*V1=C2*V2 , but there will also be a lot that dont know what the C, V stands for.. and they start making mistakes.
(a lot dont "remember" what C1 is or what C2 is...)

I'll never make that mistake, because what I do is this:

I need 3ml in total and it needs to be 5moles/ml, ok , this means I need 15 moles!
I look at my stock solution, this tells me it has 20moles/ml , but I only need 15 moles...
So I only take 0,75 ml... (because this is 15moles, I hope you know how to get this? Its a simple rule of "three", eg: 1ml ==> 20moles, Xml ===> 15moles? ) and I know I need 3ml, so what do I do? I just add more water till I reach 3 ml... And do I change the number of moles with this? NO! I have 15 moles in 0,75ml, and there will still be 15 moles in my 3ml.... (what does change? the concentration does..)

Do you see how I calculate it?

If we translate this to the formula it would be (and here most make mistakes, by not knowing what C1 or C2 means..)
C1= 20moles/ml (stock solution)
V1= how much ml I need to take from stock to archieve what I want (I want: V2 and C2)
V2= 3ml (I want 3ml)
C2= 5moles/ml

Now use this and you will see you get 0,75 too....

Using the formula is faster, but also more prone to make mistakes.....
So only use this formula if you understand (the reasoning) whats behing it!