# how can i seed 0.5 cell /well? - (Jul/16/2008 )

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hello feriends

i have a basc technique questins , i need to split my cells into 96 ell plate, seeding 0.5 cell/200 micolit media/ well? or 1 cell/well/200microlit??
my original cells density or the starter cell density is around 15x105(to the power of 5 i mean) / ml ?

-spanishflower-

QUOTE (spanishflower @ Jul 16 2008, 05:11 PM)
hello feriends

i have a basc technique questins , i need to split my cells into 96 ell plate, seeding 0.5 cell/200 micolit media/ well? or 1 cell/well/200microlit??
my original cells density or the starter cell density is around 15x105(to the power of 5 i mean) / ml ?

Is it 0.5 x 10^5 cell/200 ul/ well?

-Minnie Mouse-

QUOTE (Minnie Mouse @ Jul 16 2008, 09:14 PM)
QUOTE (spanishflower @ Jul 16 2008, 05:11 PM)
hello feriends

i have a basc technique questins , i need to split my cells into 96 ell plate, seeding 0.5 cell/200 micolit media/ well? or 1 cell/well/200microlit??
my original cells density or the starter cell density is around 15x105(to the power of 5 i mean) / ml ?

Is it 0.5 x 10^5 cell/200 ul/ well?

Hey,
I have been working with 96 well plate a lot lately (unfortunately) and I will admit I am not an expert but this is what I do and seems to work well.
Firstly, I don't really think you can plate .5cell/well. Just seems a bit strange... Half a cell??? Am I just not understanding??? Anyways your count reveals that you have 150cells/uL. The really strange thing about what your doing is the EXTREMELY low number of cells per well. Do you really want 1 cell per 200uL media? I normally put 100uL of mixed media plus cells in each well. I don't know what type of cells your using but regardless. I actually use approx 7,000 cells/well (yes, thousand) so you might want to check that. So if your count is 150cells/uL I would take my desired cell density and divide by my count (i.e. 7,000/150) and that gives you how much you need in uL per well(for 100uL total) and you merely add the remainder of media to get to 100 total uL. So everything is roughly multiplied by ten. Let me know if this helps or if this was any help whatsoever. Good luck

-WeStErNbLoT101-

well, to be a little more specified, i have a 293 stable cell line but (Bulk!!!!) i recieved it like that !!!and now i have to seed 1 cell /well in order to get a single clone and when i get it i will reseed it again in a bigger plate and check if it still contains my gene.

and if you ask how am i going to identify my positive cells? well, the gene is gfp labeled so i will check by flourescent microscopy.

-spanishflower-

If you have access to a FACS sorter, we've been sorting our GFP stables that way.

Otherwise, calculate so that you'll have 1 cell in 400 ul, then plate 200 ul per well

-Michelle4-

You absolutely cannot plate half a cell per well.. That would make no sense!!

But one cell by well is barely doable, for evident technical reasons. If you want to isolate single cells, you may want to try to plate very low number of cells in a plate, look at them in epifluorescence, and then use an isolation ring. This is a small ring that you can place on a dish (around a colony of interest) that will allow you to trypsinise it and either study it on culture it.

Okay, you can't actually put 0.5 cell per well, but what most people mean by using this is that they're sure they only have at least 1 cell per well... Usually when I used to do clones I would plate 1/2 of the 96 well plate at 0.5 cell/well and the other half w/ 1 cell per well.
Just calculate how much you need... You want 200 ul volume/well for half a plate (say 100 well iso 96 is easier w/ calculations, plus you want a little bit extra) you'll need 200 (volume) X 50 (wells) = 10,000 ul or 10 ml. In this 10 ml you want (for the 0.5 cells/well) 0.5 X 50 (wells) = 25 cells.
For 1 cells/well you'll need 50 cells. Both in a volume of 10 ml. Make sure you resuspend you cell suspension very well before counting (as I'm sure you'll know) and before you take out the (very) small volum to add to you 10 ml that you'll need for plating (putting the cells in your plate wells).
Then just add 200 ul out of the cell suspension that you made (the 10 ml) to each well.

Hope this is clear! Good luck!

-Ddkb-

I've done this... It really is as simple as you think...

Your goal, is to get a single cell in a well.... As you know, if that cell lives and devides into a nice size population, you'll have a population of cells with identical characteristics i.e. protein density, size, political aspirations.

So if you are only going to put 200 microliters of media in the cell, you 'ideally' want a density of 5 cells per milliliter, so that the chances of 200 microliters having only once cell is good...

however, some people make the cell density even lower to ENSURE the likelihood of getting only one cell in a well... This, in my opinion, is a good idea...

Sooooooooooooooooooooooo,

You want a density of 0.5 cells per 200 microliters, or 5 cells per 2 ml... OR 2500 cells per Liter.

so you collect your cells, wash them, do a cell count, and then resuspend them at a density of 10 cells per 2 ml...

You don't have to make a HUGE volume... Let's say you have 10 million cells...

- Resuspend them in 1 ml (1 million cells)
- Vortex
- place 100 micro liters of suspension in a microtube...
- Add 900 micro liters of media.

You now, have a density of 100,00 cells/ml... You can repeat the last three steps.

- Vortex
- place 100 micro liters of suspension in a microtube... (10,000 cells)
- Add 900 micro liters of media.

You now have a density of 10,000 cells per ml... and you've only used 2 ml of media... Now, you simply repeat this process 2 more times and you have 2500 cells/ml.

Of course, you can make this much faster by simply resuspending that first 1,000,000 cells in 400 L of media and this will give you that concentration of 5 cells per 2 ml...

However, Even if you found a steril flask or container that would hold 400 Liters of media, your advisor will kill you and then fire you...

-doc_t-

QUOTE (doc_t @ Jul 17 2008, 02:20 PM)
Of course, you can make this much faster by simply resuspending that first 1,000,000 cells in 400 L of media and this will give you that concentration of 5 cells per 2 ml...

However, Even if you found a steril flask or container that would hold 400 Liters of media, your advisor will kill you and then fire you...

-Minnie Mouse-

thank you all sooooooooo much
i cant really say how much i appreciate your replies, they helped me alot .

-spanishflower-

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