help needed in determining volume of cells to be seeded - (Oct/05/2011 )

Hi! my task is to passage ipsc and form EB from it.

The instructions given to me are: the number of cells to be seeded to form EB is 1X10(to the 3rd) . and the total volume/well should be 100uL.

i counted the total number of cells and its 1.115x10(to the 6th) per ml.

i did like what i did in math class: 1x10(to the 3rd) cells multiplied by ml/1115 x10(to the 3rd) =
0. 00089 ml or 0.89 uL.

For convenience purposes, i used 1uL of cell concentration and 99uL EB medium per well. and since i had to seed 8 wells; i put 8uL of cell concentration + 800uL (should be 792 but i just rounded it to 800) on a separate conical tube and then i pipette 100uL to each well.

Did i do this right? or i should be as accurate as I can when i seed cells.

Thanks a lot.

not sure what you did during math class, prop. learned some rule by hearth?

Just use logic.

You have a total of 1115x1000 cells per ml (put it in µl to make it easier: 1115 cells per µl) and in the end you need 1000 cells per 100µl.

So how do you fix this?

if you take 1µl you have 1115 cells , this is too much, you need 1000.... so how much do you need to take to have 1000 cells?

1 µl = 1115
Xµl = 1000?

==> 1/1115 X 1000 = 0,89 µl.

So just add 0,89 µl in each well and fill it up with medium/water till you reach 100µl total volume.

This is what you got: 0,89 ... (but dont use rules/trics without thinking about how they work)

but what you then describe.. I do not understand what you mean.

Lets look at what you did (if I understand it):
you took 8µl of a a cell concentrated sample (1115cells/µl), meaning you have: 8x1115 cells= 8920 cells for 8µl.

If you add these 8920 cells in 792 µl you have in the 8920 cells in 800µl (8µl+792µl) ==> 8920/800 cells per µl = 11.15 cells/µl (which you could have easly guessed because you just diluted it 100 times).

So if you take 100µl of this 11.15cells per µl solution and add this in each well, you add 1115 cells per well...
So you see.. something is off here..

I really dont understand what you are doing...
You start of good and then you do a weird thing..

Forget those mathimatical rules and trics ... use your logic, common sense... and rethink...


Or maybe you mean by "For convenience purposes" that you know you need to use 0,89 µl and that you just decided to use 1µl because thats easier (and you dont have a pippette that can handle 0.89µ?).
If this is the case: then you need to recalculate what you did and make an "in between solution" so that you can pippette a bigger volume then the 0.89 µl because if it says 1000cells perwell its 1000 cells and not 1115 !

1115 compared to 1000 is more then 10% off!!!!!!!!
This is a lot...

(depending on the experiment etc it is possible it doenst matter that much, but still: an error marge of 10% is a lot!, imagine you would get a score or 12/20 in in stead of 14/20... 10% error range on 20 ....)


+ and this is important too : "should be 792 but i just rounded it to 800" ... if you take 1µl and not 0,89 and then a bit later you also make it yoursels easier by taking 800 and not 792 ... then you are really skimming the tops off your work....

Hi Pito,

Thanks a lot for the reply.

One thing i missed to say is, i worked with a partner. All those roundin off were the suggestions, which I reluctantly followed and we worked under time pressure so after i seeded, thats when i had enough time to ponder what we did.

Anyway, next plating and seeding is independent work. So, to clarrify what u said. My first calculation was right , right? seed 0.89 uL of cells + 99.11 uL of EB medium per well.

I had to seed 8 wells, so , 7.12uL of cells + 792.88uL of EB medium would be enough and accurate to seed 100uL/well. But, for 1000uL pipetman, Im afraid 792.88 might be difficult, i guess 792.9 or 793uL.

This is what I should have followed, right?

minerva_29 on Thu Oct 6 01:09:36 2011 said:

you are right.

About the problems with 792,88 ==> yes, you need to take 793 then.

You could indeed use 7.12µl + 782.88 (or rather 783) , however it is rather a small amount, the 7.12 so you might want to work with "in between solutions" (like I allready said in my first post).
But thats up to you.
WHat I mean with "in between solutions" is the following (a simple example)=> imagine you need to dilute something 100 times you might want to do it by diluting it 10 times first and then 10 times again => add 1ml in 9ml and then add 1ml of this 10ml solution in another 9ml water => end result is 100times diluted.
Rather then taking 1ml in 99ml ... The reason is that 1ml in 99ml could give you a large error...


So its up to you to decide what you want.
Theoretically you can do it like you want, but making an "in between solution" might be helpfull.

I understood what you meant.

Thank you so much. I got more confidence now. I wasnt that confident then so it was hard to push what i thought was right.

Thanx a million!

Hi Pito,

Youve been a great help to my previous question.

Do you happen to know EB formation?

Im currently doing that and when i checked them today, EBs were formed but theyre quite small. maybe too small to be used for differentiation. what could be the reason why the EBs formed were too small?

Thanks

minerva_29 on Wed Oct 12 06:05:49 2011 said:

There could be many reasons..
But I advice you to open a new topic with this question in the right sub forum. There are others here that now more about embryoid bodies.
(you do mean embryoid bodies, right?)