Hello!
I am doing an assay with colorectal cancer cell lines using a 96-well flat-bottom plates with 1,5% agarose and it is forming what looks like spheroids. However the problem is how to remove it from the plate to an eppendorf? We tried different options like a little spoon to try to catch the agarose and hopefully the spheroid too, or to remove it with a pipette but with the pipette it was impossible because it was "stuck" in there simply.
Can anyone help me please?
Thanks in advance!
I don't know a sure answer but I would try to centrifuge the 96 wells plate upside down. In this way, you may retrieve your spheroids on the 96 wells plate lid. You may also try an agarase. Good luck
Yea try centrifugation and turning upside down plate. Also you can try serological pipettes (1.5 ml) they have larger opening and you aspirate the well content easy. Or some pipetts like these
http://www.google.rs/imgres?imgurl=https://www.sks-bottle.com/images/P200-10.jpg&imgrefurl=https://www.sks-bottle.com/340c/fin4999.html&h=350&w=254&tbnid=aBu6MvFGSnJoHM:&docid=9yNb-OS2NyV-SM&ei=OKARVqvWB8PkaIvUsJAO&tbm=isch&ved=0CBsQMygBMAFqFQoTCKvSgZjnqcgCFUMyGgodCyoM4g
I really dont know what you have in your lab from those mentioned.
I suggest to use low adherent 96 well plate instead of regular tissue culture treated well plate. you can get from any vendor like Fisher or VWR.
A better method to make spheroid is using the microwell that stem cell people use to make embryoid body. These are more expensive than normal well plate, but you can generate a lot of spheroids in short time. I use the Aggrewell from Stem Cell Technologies. They sell a 24-well plate that has 8 wells at the center modified to contain 400 microwells. So with 1 shot, you make ~400 spheroids. Link here: http://www.stemcell.com/en/Products/Popular-Product-Lines/AggreWell.aspx
This method is super easy to do to generate a lot of spheroids at once. The well plate can be reused 2-3 times by washing thoroughly with PBS after harvesting. Hope this helps.
Hi Ana,
It is best to leave the spheroids for 24-48 hrs to form after plating.
To transfer the spheroids to eppendorfs, I cut the tip of P1000 blue tip and use the P1000 pipette to suck up the contents of the well and eventually the spheroid gets into the tip. This way the spheroid does not break and you can transfer it to eppendorf.
Hi,
I normally use V shape bottom plat and didn't have any problem, cutting the tip will certainly help. Unless your agaroe is clotted, cant you start dilute the agarose with PBS until its washed away then use the pipette to retrieve your aggregates? A desktop magnifier will be very helpful.
Hi Ana,
I would use the P100 to kind of flush the bottom of the well to get them loose and then use a cut tip to collect them ( when cutting the tip I advise to use a heated knife/blade so that no pieces of plastic damage the spheroids).As mentioned, you can try to use microwells, the Agrewell dishes mentioned above are quite expensive so I am trying to use them as molds to make them out of agarose (see publication http://www.sciencedirect.com/science/article/pii/S0142961212014044). This will be better than an ultra low attachment 96-well plate since there you have no control of the initial number of cells to form 1 spheroid neither ensure that only 1 will be formed per well.
Good luck!
Hi Ana,
Very nice answers from all the previous writers.
Just one comment, if you do not have aggrewell available, you can use the older method of hanging droplets to generate spheroids in suspension which, may require a bit of practice, but I'l say it is definitely worth a try. For more information on the hanging droplet methodology, you can look at this video article on pubmed central: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3197119/
I hope that helps out :)
Good luck !!!
Udi
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