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Cell counts for Clonogenic and MTT assay

Clonogenic MTT

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#1 SteadyLearning

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Posted 14 July 2014 - 05:41 AM

Hello guys,

 

I have to do a clonogenic and a mtt assay to test the toxicity of two different drugs accross three cell lines. Im struggling to get to grips with determining the cell numbers to plate out initially before any drug is added to the plates.as well as how to lay these out (kind of left to get on with it and it is my first time doing both assays sad.png ) different protocols online are really confusing me so I am sorry in advance for any noob questions of anything I may have overlooked. 

 

So, after i have trypsinized my cells and it is time to resuspend my pellet, how much media do i resuspend in? or does this not matter? and once I have my cell count, do I treat my re-suspended pellet suspension as my cell stock, and take however many cells I need from there?

 

Lets say I want to put a 1000 cells in each well (of a 96 well plate), to work out how much I need to take from my stock do i divide the number of cells I want over the number of cells I have counted and mulitply this by the volume I resuspended in followed my x by 1000 to get it into ul? 

 

Any help or advice will be greatly appreciated

 

Many thanks



#2 bob1

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Posted 14 July 2014 - 12:40 PM

So, after i have trypsinized my cells and it is time to resuspend my pellet, how much media do i resuspend in? or does this not matter? and once I have my cell count, do I treat my re-suspended pellet suspension as my cell stock, and take however many cells I need from there?

 

Lets say I want to put a 1000 cells in each well (of a 96 well plate), to work out how much I need to take from my stock do i divide the number of cells I want over the number of cells I have counted and mulitply this by the volume I resuspended in followed my x by 1000 to get it into ul?

The volume you use doesn't matter.  However, if you add too much medium then your cells will be too dilute to give an accurate count, and too little will make them too concentrated to give an accurate count.    Once you have your count, your resuspended cells can be considered a stock if you like, or you can re-pellet and resuspend at a convenient concentration if you prefer (which is what I usually do).  Either way you use the same formula to work out how many cells you need for the task in hand.  What I would recommend doing is calculating the total cell number that you have in your pellet (n=C*V) and using this to make your dilutions from a defined concentration - it makes your calculations easier for you to look at and work out if you have the calculations wrong.

 

Just like you would have been taught in school, it is a good idea if you write out what you want to achieve, the formula you are going to use and the variables that you have.

 

In many instances the formula will be: C1V1=C2V2,  this is used to get from one concentration in one volume to another concentration in another volume.  For example if you have 10^6 cells/ml (C1) and you want 1000 cells/ml (C2) in a total volume of 10 ml (V2) then you have (1000 * 10)/10^6 = 0.01 ml (10 ul) as the value for V1.  Note that pipetting 10 ul of cells is usually inaccurate as they settle quite fast, so it would be a good idea to dilute your initial suspension some (maybe 1:10) so that you are pipetting a bigger volume.



#3 SteadyLearning

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Posted 15 July 2014 - 12:12 PM

^^^ Thank you very much biggrin.png

 

 



#4 bob1

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Posted 19 July 2014 - 01:53 PM

Sent Yesterday, 06:35 PM

Hey, So I followed your advice, and my cell counts worked perfectly! Thank you again, you have been a tremendous help! 
 
If I could ask you about my clonogenic assay please, as i will be using radiating my cells from 0-5Gy's. I need to increase the amount of cells i have per 6 well plate right (according to the plating efficiencies of my cells)
 
for my V79 cells the PF is 80%, AGO cells 60-70% and LN18 cells the PF is around 3%. 
 
Say i will add 2 ml of media to each of the 6 wells and i want 100 cells per well for the 0Gy radiation, 200 for 1Gy and so on. do i use the same formula we discussed for the MTT assay? I am confusing myself as to how to work out how much of my pelleted cells cells to add to a total volume 7ml of media, and then take 2 ml of this 7ml and add across the 3 wells for each radiation dose. 
 
Thanks in advance for your guidance


What I would do is calculate the amount required for your highest concentration at 2 ml, then work out how much of that to add to each well for lower concentrations, then top up with medium. For example, if I wanted 1000 cells/well in 2 ml (500/ml) for my highest and 200/well for my lowest, I would need to add V=N/C V=200/500 = 0.4 ml and add 1.6 ml medium.




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