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96 Well Edge Effect


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#1 Tensix

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Posted 25 July 2013 - 11:14 AM

I've been seeding cells in 96 well plates for drug sensitivity experiments.

In the protocol I'm following, I seed the 60 wells from B2-H7, and each column is 6 replicates of a single drug concentration or control. The wells surrounding these 60 are filled with PBS and unused to avoid anything screwy happening with evaporation.

Despite this, I noticed this morning that all of the outermost used wells have a clear edge effect. Cells in these wells are heavily concentrated on the edge of the well facing outwards, nearest the PBS. This effect is consistent over 10 plates with 2 different cell lines on different shelves of the incubator.

I've read online that some companies sell plates designed to avoid this kind of thing, but is there anything I can try in the meantime to avoid this?

#2 x26s

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Posted 25 July 2013 - 12:42 PM

Try not using pbs at all!
The key to your solution might lie in the answer to the question: why did the cells face the pbs?
Best of Luck!

Edit: Also try using the same content as that of B2-H7 wells (media+cells+drugs) in the outer wells as well, if you don't run short of it, or just the media.

Edited by x26s, 25 July 2013 - 12:56 PM.


#3 bsmith

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Posted 12 August 2013 - 07:44 PM

Hope you're not still having this problem, Just in case

 

Plating some wells of Media/PBS/whatever around the wells containing your cells isn't the only way to reduce edge effect. If the PBS isn't the same temperature as the cell suspension that you are adding to the wells, you will create a thermal gradient across the well. i.e. hotter or cooler in the outside wells which are nearer to the PBS.

 

Instead, once you are done plating. Do not shove the cells straight in the incubator (37 C), allow 1 hour incubation at room temperature (22 C or whatever) to allow cells to settle evenly in all the wells. And if you do want to stop evaporation, make sure your PBS and cell suspension are at similar temperatures. Better yet, let the cells settle for an hour, then add the PBS to the surrounding wells. 

 

"A simple technique for reducing edge effect in cell-based assays" by Lundholt et al. in 2003 (PMID: 14567784) explains this process.



#4 Tensix

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Posted 30 August 2013 - 11:29 AM

Hope you're not still having this problem, Just in case

 

Plating some wells of Media/PBS/whatever around the wells containing your cells isn't the only way to reduce edge effect. If the PBS isn't the same temperature as the cell suspension that you are adding to the wells, you will create a thermal gradient across the well. i.e. hotter or cooler in the outside wells which are nearer to the PBS.

 

Instead, once you are done plating. Do not shove the cells straight in the incubator (37 C), allow 1 hour incubation at room temperature (22 C or whatever) to allow cells to settle evenly in all the wells. And if you do want to stop evaporation, make sure your PBS and cell suspension are at similar temperatures. Better yet, let the cells settle for an hour, then add the PBS to the surrounding wells. 

 

"A simple technique for reducing edge effect in cell-based assays" by Lundholt et al. in 2003 (PMID: 14567784) explains this process.

 

Interesting.  The temperature of the PBS was definitely something I hadn't considered.

 

I'll try the hour incubation and then add RT PBS today and see how it goes.

 

Thanks!






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