Seeding plates - (May/06/2011 )
pito on Sun May 8 08:02:51 2011 said:
bob1 on Sun May 8 07:55:01 2011 said:
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.
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 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.
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!
...nice logic, pito....actually, this very common mistake in calculating the number of cells needed happen when we start mixing total amount or number of cells and concentration. But really, I agree, 6 million per well is a lot esp if it is just for seeding which normally implies that you'd still wait for the cells to grow, propagate or become confluent etc. So it's most probably 6 million for the whole 12-well plate...but..protocols are seldom given like this....it's usually how many cells you need per well...hence the confusion.
Hey kumara...come back in here and clear this matter up....
-casandra-
casandra on Sun May 8 10:47:00 2011 said:
...nice logic, pito....actually, this very common mistake in calculating the number of cells needed happen when we start mixing total amount or number of cells and concentration. But really, I agree, 6 million per well is a lot esp if it is just for seeding which normally implies that you'd still wait for the cells to grow, propagate or become confluent etc. So it's most probably 6 million for the whole 12-well plate...but..protocols are seldom given like this....it's usually how many cells you need per well...hence the confusion.
Hey kumara...come back in here and clear this matter up....
You are right, but I am not sure its a "practical" problem here... since someone else asked the same question, I am starting to think its a theoretical example just for the calculation of it... and often in those examples they use weird numbers.
Or maybe its even made especially to see if they understand why it makes no sense? Eg: they indeed need to calculate thus, but the smart ones would thing and reply: yes, its Xmil cells, but can we put so many cells in a well....
-pito-
Hey guys i thank each and everyone who replied to my question.
I am sorry too in not replying to you guys as I am on holidays for these few days.
Anyway i am back now and happy to see your replies.
I think I confused you all with my question.
In a hurry i did a mistake in my question.Sorry again
Number of cells is 24*10000 cells/ml
the change is like i need to seed in 12 wells of a 6 well plate( i mean 2 sets of 6 well plates, one set for sample(6 wells) and one set for control(6 well)) where in each well I need to have 6250000 cells in a total volume of 2.5ml per well
hope it is clear now
Please help me.
-harry348-
harry348 on Wed May 11 14:22:11 2011 said:
Hey guys i thank each and everyone who replied to my question.
I am sorry too in not replying to you guys as I am on holidays for these few days.
Anyway i am back now and happy to see your replies.
I think I confused you all with my question.
In a hurry i did a mistake in my question.Sorry again
Number of cells is 24*10000 cells/ml
the change is like i need to seed in 12 wells of a 6 well plate( i mean 2 sets of 6 well plates, one set for sample(6 wells) and one set for control(6 well)) where in each well I need to have 6250000 cells in a total volume of 2.5ml per well
hope it is clear now
Please help me.
You need 6.250.000 cells per well but you only have 240.000 cells/ml in 10 ml, thus 2.400.000 cells in total......
-pito-
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-harry348-
harry348 on Wed May 11 14:46:03 2011 said:
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are you sure your info is right?
-pito-
harry348 on Wed May 11 14:46:03 2011 said:
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What do you mean blank?
Btw, are you and kumara one and the same? Will you double-check again the number of cells you need bec 6.25 million per well seems pretty steep (perhaps it's one too many zeroes?)...anyways, just do a rough calculation....you need about 75 million cells i.e. 2.5 mill/ml X 30 ml (2.5 ml X 12 wells) or 6.25 mill X 12 wells...and you only have about 5 million cells in total (240,000 cells X 20 ml) kumara did write 20 ml or like pito wrote only 2.4 million if you only have 10 ml...are you sure that your stock is only this much (one or two less zeroes)? You may have to expand them more if you really need the 6.25 million per well... -casandra-
I resuspended the pellet in 20 ml medium and in this i got around 24*10000 as the number of cells per ml
-harry348-
harry348 on Wed May 11 15:07:14 2011 said:
I resuspended the pellet in 20 ml medium and in this i got around 24*10000 as the number of cells per ml
so you only have 4.8 million cells in total......therefore, if your numbers are all correct, you don't even have enough cells for one well.....
-casandra-
If I remember correctly, you once wrote 625.000 cells per well in another post not 6mil....
(use . between your numbers, its easier to read and you would make less mistaks, because now I have to idea you made typing errors in the numbers and you dont even know anymore what it has to be...)
-pito-