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cell count - (Jul/01/2011 )

I am growing some cells in 96 well plate. My cells are in 200ul total solution in each well. I did a cell count an it 7X10^6 cells. But I guess that is 7X10^6 cells/ml. So, to know how many cells i have in the well, is it 1.5X10^6 cells?

Also, I am growing cells in 2ml wells. If the same count is for that per ml, does tat mean it is (7X10^6 cells ) X 2 for cells in that well?

-Funny-

Funny on Fri Jul 1 19:42:05 2011 said:


I am growing some cells in 96 well plate. My cells are in 200ul total solution in each well. I did a cell count an it 7X10^6 cells. But I guess that is 7X10^6 cells/ml. So, to know how many cells i have in the well, is it 1.5X10^6 cells?

Also, I am growing cells in 2ml wells. If the same count is for that per ml, does tat mean it is (7X10^6 cells ) X 2 for cells in that well?


You said you did a cell count.. but a cell count in what?

We need to know in what volume you did that cellcount...
There is no way to know what the cell count is if you dont know what the volume was that you counted.

-pito-

I did a cell count 1:20 dilution in total 200 ul(10ul of my cells + 190 ul of trypan blue). 10ul of the cells I took was from 200ul culture in a 96 plate well

-Funny-

So you counted your cells and did the calculations... can you show the calculations so that we can see what you have done. This will tell us whether you have a total number of cells or a concentration.

-bob1-

sure. I took 10ul of my cells growing in 200ul wells and added 190 ul of trypan blue. counted the cells in four outer squares. then the total no. of cells X 20 (dilution factor) X 2500 was my cells per ml.

Is that not right?

-Funny-

Looks OK, though I do it a different way - each larger square on a haemocytometer (usually there are 9 of these, divided into 16 or 25 smaller squares and bounded by 3 lines) is 1/10000 of a ml. So to count your cells you need to count more than 1 square, preferably at least 2 per side (error is +/- 15% - if your counts are not within this error you should repeat), divide by the number of squares you counted, multiply by the dilution factor and add 1x10^4 on the end. This will give you the cells/ml.

To get total cells multiply by the volume in ml.

1.5x10^6 cells is an awful lot for each well of a 96 well plate - your cells are extremely high density which will be affecting how they behave. Typically you would expect approx 5x10^4 cells maximum out of a well of a 96 well plate, assuming confluent growth.

-bob1-

Thanks for that. Just clarifying, the cells we count by hemocytometer is the cells/ml? So, we can calculate the total number of cells. Suppose in my case, the number of cells counted is in per ml, then I have to calculate no. of cells in 200ul (since they are growing in 96 well plates with 200ul in each well). This is will give me cells per well.

-Funny-

Funny on Sun Jul 3 21:09:32 2011 said:


Thanks for that. Just clarifying, the cells we count by hemocytometer is the cells/ml? So, we can calculate the total number of cells. Suppose in my case, the number of cells counted is in per ml, then I have to calculate no. of cells in 200ul (since they are growing in 96 well plates with 200ul in each well). This is will give me cells per well.


yes.

You just need to "recalculate" it so you get a #cells per 200Ál

If you know how many cells there are in 1 ml, its easy to know how many there are in 200Ál and if a well is 200Ál then yeah, just calculate it for 200Ál.

You should look up how a hemocytometer works and how they come to that certain volume... once you undesrtand this (its basic math) you will never need to worry about calculations like this again....

I advice you to check it, see if you understand the concept.

I have the feeling you think that the 1ml is a fixed numbr or something that "magically" comes from somewhere and is a general rule.. its not....

Think about what a cube is, what is the volume of a cube? What is the shape of those cells in a hemocytometer?

-pito-

Thanks. I get it now. I will look more into it.
Thanks

-Funny-