[nealsynter (OP)]

Hi Guys-

I'm new to Tissue culture and have a basic question.

I have 2 cell lines: Line X doubling time=50 hr and Line Y doubling time=37 hrs.



My question is that I want to do a 5 day tmnt with a drug and harvest all the samples at the same time. So for example, I'll start my 5 day tmnt. on a Fri etc--see below. And harvest all the samples on the following Wed. My issue is that I need to maintain similar density for ALL samples--how do I calculate what density I should plate all the tmnt samples so that when I harvest cells on Wed--the data won't be confounded by cell density? Thanks so much!!!!






Day   Tmnt.Time   Start tmnt
Fri   d0   5d   12pm
Sun   d2   3d   12pm
Tues   d4   1d   12pm
Wed   d5   3hr   9am
Wed   d5   Ohr   12pm

[Eternal Student]

Sorry,   I'm not familiar with the acronym  tmnt.    Google just keeps telling me about  Teenage Mutant Ninja Turtles.
    I'm suspicious there's some maths to be done here but it might take too long to explain what tmnt means.   I'll leave this for others to answer.

Best Wishes.

[evan_au]

"tmnt" = "treatment", with a drug

Note that we are not here to do your homework!
- But we can give some hints if you are stuck...

One practical way to do it is on a spreadsheet:
- Add up the hours for doubling time, and find out how many doublings there are in 5 days = 120 hours:
- Line X: 120/50=2.4 doublings 
- Line Y: 120/37 = 3.2 doublings

Then allow enough space for the growth given the final number of cells.

maths_in_workplace.jpg (48.57 kB . 843x843 - viewed 2607 times)
To do it properly, use logs and exponentials.
To do it even more properly, use differential equations...

[nealsynter (OP)]

Thanks a lot! OK so I get the fact that Cell X will have 2.4 doublings and Cell Y will have 3.2 doublings

From here I'm lost! What's the equation that I would need to use to figure out the plating cell # that takes into account the # of doublings, time in culture etc! That's what I'm confused about! This is what happens when you graduate from a community college in the US!

[Eternal Student]

Hi again.

A fair amount of time has passed and there's been no additional replies, so I'll try and say a bit more.

From here I'm lost!

   Well I'm not even sure I understood the question.

I think this is what you were doing:
You prepare a total of  8 different plates with some cells you are culturing.    One each of Line X and Line Y on the days   Friday, Sunday, Tuesday at the time of 12 pm   (which is midday).   The last two plates are prepared on a Wednesday at 9 am.

This gives the cultures on the plates a different amount of time to grow.

You say something about not wanting cell density to confound the results but said nothing about what tests you were trying to perform or what those "results" might be.    You could be performing a test where you just select one cell from every plate and then the final number of cells on each plate is irrelevant anyway.  I'm guessing you're doing something where you would want every plate to end up with the same number of cells,  N,   when you finally harvest all the samples.

Anyway, if that's what you were doing, then you need to know this formula:

      N = Number of cells you finish with =    N₀  . 2 ^t/λ

[Equation 1]

where  N₀   = the number of cells you started with.
  t=  the number of hours that have passed.        λ = the doubling time (in hours).

 Evan_au  was suggesting you just use something like a spreadsheet to try a few  initial conditions   (some  N₀ ) values   that will end up giving the same final number N for the number of cells you finish with in each plate.   For a better result just actually do the mathematics.   To be honest, I think it would take longer to use a spreadsheet.

    I don't suppose you really need to derive [Equation 1] from first principles, just to be able to use it.   If you do want to derive the equation, great... and there are many references for that you could look at.

    There's also a lack of information about how many cells a plate can support before you consider it to be overcrowded.   Also there's usually some nutrients in the medium of the plates that can become depleted if you tried to sustain too many cells on it for too long.   Overall, you haven't presented enough information and we just have to guess what the final number, N, of cells that you end up with on the plates should be.  A reasonable guess might be 400.  Either specify the value of the number of cells you want to end up with (the N value)   OR ELSE   you will only be able to determine the initial number of cells that should have been plated as a fraction or proportion of   N.

Best Wishes.

[nealsynter (OP)]

Thank you so much! I really appreciate it!
So one thing that's challenging to me is that these are suspension lines. I just need to plate them at an initial density that is not too low so that they won't proliferate. I'll be submitting them for proteomics and was told to make sure that the concentration for treated and untreated cells are fairly close when I harvest all the cells on Wed.

So I'll use the formula you suggest: Total # cells= Starting cell # * 2^ hrs. passed/doubling time
Lets say I want 4 million per ml for all the samples. How would I begin!
My attempt: 4,000,000= 2 to the power of 120/70. This would be for the 5 day? Can you check my calculation on this.
and then I'll subsequently do 4,000,000=2 to the power of 72/70 for the 3 day?

Thanks so much!!!

[Eternal Student]

Hi.

    It sounds reasonable but there's a   symbol   missing  ( N₀  ) and the number 70 seems to have come from somewhere instead of using the doubling times you originally stated.

You would set:
   4 000 000 =   N₀   .  2^(120/50)     for  the  5-day sample of cell line X.    Note that this had a doubling time of 50 hours  (not 70)   and the  unknown  quantity  N₀ = the initial concentration you want to plate up, does appear.

    You would re-arrange that formula to find  N₀,    then grab a calculator and calculate it.
N₀   =   4 000 000     /    (2^2.4)           ≈        757 858

So, for the culture from cell line X, which was left to grow for the full 5 days,   you  start by plating up a concentration of approximately 757 thousand cells per ml.

    For the cells from line Y,  left to grow for the full 5 days.   You follow the same idea....
Set    4 000 000  =   N₀  .  2^120/37         .   Note that I have replaced the 50 with 37 because these cells double every 37 hours.   The 120 stays because this culture is still left to grow for 120 hours.
   Re-arrange this to find   N₀.


For the 3-day sample,  you do something similar remembering to change the 120 hours  for  72 hours   AND  also replace the doubling time with 50 or 37  appropriately.
Do the calculation again for 1-day, both cell lines and get their initial concentrations.    Do once more for the 3 hr. both cell lines.

      You should always be doing this final calculation:
    N₀    =   4 000 000   /    2^X         Where   X =    time left to grow (in hours)  /   doubling time (in hours)

If I've understood correctly, you have  4 different times   and   2 different cell lines each time,   so you should calculate 8 different  values of   N₀.         Those  values are determined by sticking in the appropriate different combinations    of   (the time left to grow)   and the  (doubling time).

Best Wishes.

[nealsynter (OP)]

Thank you so much!!! I totally get it!!!!!

[set fair]

If maths is not your strong point, then the easy way is to just plot the density of the two cultures over time on two graphs. but as Evan implys it's blerry obvious that you asking for help with your homework, you're not a good liar.