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Cell counting

cell count cell counting hematocytometer

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11 replies to this topic

#1 Mad Researcher

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Posted 14 November 2018 - 05:26 PM

Hi,

 

I plated my cells at a density of 50,000 cells/well in 1 ml of media.

 

I have treated my cells with drugs and have counted the cells. I wanted to know the cell density after drug treatment, how can I calculate that?

 

Thanks


Cheers,

Mad Researcher

#2 bob1

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Posted 14 November 2018 - 07:06 PM

Density is cells per unit area. If you know the area of the wells you had your cells in, then it is simply:

 

number of cells/area of well.

 

The technical documentation for the plates you have usually tells you area. Otherwise the Roche Life Sciences FAQs has a table with usual values.



#3 Mad Researcher

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Posted 14 November 2018 - 07:13 PM

Density is cells per unit area. If you know the area of the wells you had your cells in, then it is simply:
?
number of cells/area of well.
?
The technical documentation for the plates you have usually tells you area. Otherwise the Roche Life Sciences FAQs has a table with usual values.


For example if my cell count was 500 and my plate is 1.8 cm^2, then It would be 277. What would be the unit and should I consider the dilution factor of 1:1 as I used trypan blue
Cheers,

Mad Researcher

#4 bob1

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Posted 14 November 2018 - 07:43 PM

The unit is cells per area - this could be whatever unit area you want, but it would be typically be cm2 or mm2. It would be written cells/cm2 for example.

You would need to calculate the total number of cells per well. Assuming you are using a hemocytometer:

 

1)count number of cells in the 9 big squares (bounded by 3 lines in most modern hemocytometers)

2)divide that number by 9 (to get cells per one square -each square has a defined volume of 0.1 mm3)

3)Multiply by dilution factor

4) multiply by 10000 to get cells per ml

5)multiply by the volume you collected your cells in.

 

For an example. If you have 500 cells per side (9 big squares) of the hemocytomter, and you collected your cells in 3 ml and diluted the count 1:1 with trypan blue.

 

1) 500

2) 500/9 = 55.555 cells per square (0.1 mm3)

3) 55.555 *2  = 111.111 cells per square

4) 111.111 * 10000 = 1111110 cells per ml

5) 1111110 * 3 = 3333330 cells total

 

Now divide by the area of the well (1.8 cm2 in your case)

3333330/1.8 = 1851850 cells/cm2



#5 Mad Researcher

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Posted 30 November 2018 - 07:49 AM

The unit is cells per area - this could be whatever unit area you want, but it would be typically be cm2 or mm2. It would be written cells/cm2 for example.

You would need to calculate the total number of cells per well. Assuming you are using a hemocytometer:

 

1)count number of cells in the 9 big squares (bounded by 3 lines in most modern hemocytometers)

2)divide that number by 9 (to get cells per one square -each square has a defined volume of 0.1 mm3)

3)Multiply by dilution factor

4) multiply by 10000 to get cells per ml

5)multiply by the volume you collected your cells in.

 

For an example. If you have 500 cells per side (9 big squares) of the hemocytomter, and you collected your cells in 3 ml and diluted the count 1:1 with trypan blue.

 

1) 500

2) 500/9 = 55.555 cells per square (0.1 mm3)

3) 55.555 *2  = 111.111 cells per square

4) 111.111 * 10000 = 1111110 cells per ml

5) 1111110 * 3 = 3333330 cells total

 

Now divide by the area of the well (1.8 cm2 in your case)

3333330/1.8 = 1851850 cells/cm2

Hi again,

 

There is some confusion in my cell counting. I plated 50,000 cells/well.

After drug treatment, I found that  I have 39+45+40+48 cells in the 4 quardants. So, I divided the total by 4 i.e. 172/4 multiplied by 2 and then 10,000, which gave me 3440000 cells in total. My final volume was 1. After I divide it by 1.8 cm2, I still get 1911111.11 cells/cm2 which is way too much then I plated. 

Can you please tell me what am I doing wrong here?

 

Also, why do you divide by 9? I count the 4 big squares so, I divided by 4.

 

Thank you.


Cheers,

Mad Researcher

#6 bob1

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Posted 30 November 2018 - 08:52 AM

You plated 50,000 cells per well and they grew is the simplest explanation. Another potential explanation is that you seeded more cells than you thought - if you left the tube to sit, even for a minute, before taking the counting aliquot then more than half of the cells in there had settled and were likely not included in the count, so your calculations would be off significantly. If the cells are clumped at all this increases the error in the count, it is hard to count the number of cells in clumps and they settle, resuspend and distribute in the well differently to single cells.

 

It is also well known that even with an experienced user highly consistent counting with a hemocytometer is difficult and has a minimum error of +/-15%

 

So - some questions:

 

1) Did you use trypan blue for your cell counting, if so, how much did you add to what volume of cells?

2) How did you arrive at the 50,000 cells per well - can you show your calculation?

 

I don't see how you got 3.44 million (3,440,000) in your calculation. By my calculation: 172/4*2 (or 172/2 to simplify) is 860,000 - still way more (17x) than you plated! 

 

I used 9 as there are 9 large squares (3x3) on each half (1 grid) of the hemocytometer. You counting 4 is fine too, but it increases your potential error. You must use a single cell suspension, count at least 100 cells per side, and you should load and count both sides, then average results to get the most accurate counts. 



#7 Mad Researcher

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Posted 30 November 2018 - 09:21 AM

You plated 50,000 cells per well and they grew is the simplest explanation. Another potential explanation is that you seeded more cells than you thought - if you left the tube to sit, even for a minute, before taking the counting aliquot then more than half of the cells in there had settled and were likely not included in the count, so your calculations would be off significantly. If the cells are clumped at all this increases the error in the count, it is hard to count the number of cells in clumps and they settle, resuspend and distribute in the well differently to single cells.

 

It is also well known that even with an experienced user highly consistent counting with a hemocytometer is difficult and has a minimum error of +/-15%

 

So - some questions:

 

1) Did you use trypan blue for your cell counting, if so, how much did you add to what volume of cells?

2) How did you arrive at the 50,000 cells per well - can you show your calculation?

 

I don't see how you got 3.44 million (3,440,000) in your calculation. By my calculation: 172/4*2 (or 172/2 to simplify) is 860,000 - still way more (17x) than you plated! 

 

I used 9 as there are 9 large squares (3x3) on each half (1 grid) of the hemocytometer. You counting 4 is fine too, but it increases your potential error. You must use a single cell suspension, count at least 100 cells per side, and you should load and count both sides, then average results to get the most accurate counts. 

Thank you for your prompt response.

 

1) I use 50 µl of cell suspension and 50 µl of Trypan blue.

2) My calculation for cell plating is:

Counted cells - 632

Squares - 4

Dilution - 2

So, I had 3160000 cells/ml

 

I made a stock conc. of 100,000 cells/ml and diluted it to 50,000 cells/well. 

 

It maybe my calculation was wrong. If it is, could you please help me with the right calculation.

 

Also, by treating the cells with drug, I was expecting less cells, as the drugs will kill some cells out of 50,000 I plated.


Cheers,

Mad Researcher

#8 bob1

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Posted 30 November 2018 - 11:57 AM

That looks like the correct calculation. The dilution factor was correct too.

 

I presume you seeded the cells then let them sit for a while, perhaps 24h  before treating with the drug? If so, the cells, depending on which cell line you are using, may have undergone between 1 and 3 replication cycles. If you assume 3 cycles the equation is 50000*2*2 = 200000. Exponential growth...

 

Now assuming your drug treatment is on for another day and kills 50% of the newly divided cells at each division cycle (brackets represent each division cycle) - (200000*1.5) + (300000 *1.5) + (450000 *1.5) = 1425000 cells/well or 28.5 x the number you put in.

 

Now I am aware there is something wrong with how I have explained that, relating to the replication and killing 50% - me not math good, but I am sure you get the general idea.



#9 Mad Researcher

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Posted 30 November 2018 - 12:02 PM

That looks like the correct calculation. The dilution factor was correct too.

 

I presume you seeded the cells then let them sit for a while, perhaps 24h  before treating with the drug? If so, the cells, depending on which cell line you are using, may have undergone between 1 and 3 replication cycles. If you assume 3 cycles the equation is 50000*2*2 = 200000. Exponential growth...

 

Now assuming your drug treatment is on for another day and kills 50% of the cells at each division cycle (brackets represent each division cycle) - (200000+100000) + (300000 +150000) + (450000 +225000) = 1425000 cells/well or 28.5 x the number you put in.

Thanks for the explanation.

 

I am plating primary neurons from the cortex. And I treated the cells after 10 days of plating. Does that make a difference?


Cheers,

Mad Researcher

#10 bob1

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Posted 30 November 2018 - 12:11 PM

Primary neurons will grow quite slowly I would assume, probably slower than one replication in 24 h - if you know the doubling time, you could do the calculations yourself. I was assuming cultured cell lines rather than primary cells, so the replication figures I gave above don't really apply. Yes, the plating time makes a difference - the longer you give them, the more they will divide. However, I know primary neurons are slow growing and susceptible to damage fairly easily so you want to give them some time to recover after plating before you start drug treatment.

 

Glial cells replicate quite fast, so it could be that you have some glia in there which are replicating more rapidly. 



#11 Mad Researcher

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Posted 30 November 2018 - 12:14 PM

Primary neurons will grow quite slowly I would assume, probably slower than one replication in 24 h - if you know the doubling time, you could do the calculations yourself. I was assuming cultured cell lines rather than primary cells, so the replication figures I gave above don't really apply. Yes, the plating time makes a difference - the longer you give them, the more they will divide. However, I know primary neurons are slow growing and susceptible to damage fairly easily so you want to give them some time to recover after plating before you start drug treatment.

 

Glial cells replicate quite fast, so it could be that you have some glia in there which are replicating more rapidly. 

Thank you. It might be 10 days was too much for them, I could have started earlier. 

This was a mixed culture, so there must be Glial cells in them. Do you know how fast the Glial cells replicate?


Cheers,

Mad Researcher

#12 bob1

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Posted 30 November 2018 - 01:05 PM

A quick search of the literature tells me about 20-24 hours per replication cycle for glial cells from neonates, same time for astrocytes too. It looks like neural progenitor cells replicate faster than that 13-17h/cycle according to one paper I found.







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