upscaling to larger dish - (Sep/01/2011 )

Hi all,

I have encountered another issue that's been mind-boggling to me. I am trying to upscale my experiment from 24 well to the 100 x 20 mm dish, since I did a screening of activity in the 24 well but then wouldn't have enough protein for western blotting. I therefore needed to upscale the experiment to large dishes.

Does anyone know if when you upscale the experiment, do you upscale to make sure that the concentration is adjusted based on cm2 or per consistent amount of cells per well or any other criteria? I noticed that even when I upscale the cell density based on per consistent cm2, the cells did not behave similarly as when it was in the 24 well (e.g. it went to confluence really quick in 24 well but not in the 100 x 20 mm dish).

If anyone has any insights..

Presuming you mean cells/cm² for this one, confluency is based on cells per area, so that is how it is normally done for scaling the plating.

Cells can behave differently if they are being plated onto different surfaces or in larger volumes relative to what you would with the 24 well plate (e.g. using 1.0 ml for the well compared to 0.5 ml), as this dilutes out growth factors that the cells produce, which help other cells to grow.

You may find it easier to scale up to wells of a 6 well plate - 2 wells is usually heaps for a western.

Hi Bob1, thank you for the response. I am however also wondering of how do you upscale the concentration of the compound.. I am completely confounded as I found that in 24 wells my compound would promote cytokine release and when i upscale to large dishes, I adjust for absolute amount of compound per cm2, and my cytokine was suppressed instead.

Typically compounds are based on a concentration - e.g. mg/ml or mol/L not on area, which is probably your problem, as you are unlikely to be scaling the amount of diluent (e.g. medium or PBS) based on area (e.g. I would use 0.5 ml for a well of a 24 well plate (2 cm²) but would only use 10 ml or less for a 100 mm dish (56 cm²), not 23 ml as scaling would suggest).

Thank you Bob1. I think I will try the 6 well idea. Nevertheless, I was thinking about it and wondering, if you upscale the cell number and adjust it per cm2, but then upscale the compound with same concentration but with 10 ml of diluent instead of upscaling the amount of diluent as well, wouldn't that mean that there is generally less compound exposed to cell or per cm2?

No, concentration is concentration, the total amount will be less, but the amount per volume will be the same so the exposure should be identical as volume is just "area x height".

Thanks Bob1. I am trying out the six well plates as well as the 24 well plates, and few large dishes to compare if I can get a similar trend in the six well or the large dishes.

Hi Bob1, so I finally tried out the six well plates and it did produce the trend that is similar to that from 24 well in term of cytokine release. Then I tried scraping it off for the nuclear extract to be extracted. Unfortunately I could only obtain about 1/3 the amount of protein that I could get from large dishes (even from collecting from two wells). I am just wondering, do you happen to know if it's a common practice to pool even three or four 6 well plates together?

Absolutely, I know people who pool more than 6 wells per treatment sometimes.

Oh boy. ok. The problem is just that I have 3 compounds at 3 concentrations with one negative and one positive control in triplicate, and potentially a couple of different time points. I was worried that more wells = more time scraping. WIth more time sensitive transcription factor I worry I might miss the 'moment' where the protein is in the nuclear fraction between the first well to the last well scraped = not exactly a reliable data. What I do then usually is that I removed all my culture media and put everything to the fridge while scraping what I have on hand. I am wondering if you have suggestions as to how to approach this better?

I am thinking now even to narrow down my concentration/compounds to those that are relevant to my other set of experiments.. may be in depth information of specific compound/concentration is better than breadth of information on compounds/concentration that likely would not have effects anyway. If you have opinions on this, I would appreciate it too.