Hi! Does anyone have a tip for a dissociation agent that isn't Trypsin-EDTA or affect membrane receptors? Trypsin cleaves our membrane receptors. I use QBIHEK293A cells. They are like HEK293 cells, but more adherent. I have used Versene (EDTA-solution) for 10 min at 37 degrees, and they will not detach. I've tried scraping after 10 min at 37 degree, and it worked so and so, but I have a big problem with clumps. Since I'm doing a cell based assay with living cells, and I need to count them before I start, this is a huge problem. I see other dissociation agents are available, but I'm not familiar with these, so I was wondering if any of you had some tips or have found a solution to a similar problem? Many thanks for your help!
Hi Henriete when we don't use trypsin we tend to use Accutase (also an EDTA solution)
http://www.sigmaaldrich.com/catalog/product/sigma/a6964?lang=en®ion=GB&gclid=CJGaq725wdACFcO4Gwod0BUDHw
However be aware that Accutase (and I assume Versene) is best used at room temperature - placing it at 37oC will actually inactivate it (i.e. don't need to add FBS media to inactivate as you do with Trypsin). You might want to check with your Versene protocol in case this too is best used at room temperature.
Unfortunately some cells are hard to detach (the worst time I have had were with the LS147T cells).
For these I have to add Accutase, scrape, leave in Accutase (room temp) and re-scrape followed by trituration 2-3x (mixing thoroughly by pipetting).
Pipetting the cells up and down 2-3x with a (i.e.) 10 ml pipette (with the tip pressed flat against the bottom of the flask) will help break up clumps but can be damaging to the membranes of more delicate cells.
Sorry I can't be ore help,
Gary
I use Accutase from stem cell and it work fine to dissociate iPSC at 37°c for less than 5 min. I use also dispase for recovery of uortheliale cells without a major problem
Please try them and let us know
Good a luck
Hello Henriette
I have experience with 0.53mM EDTA/PBS at 37ºC and 10 minuts. However, you can try detach the cells with rubber policeman.
Regards
Thank you for all your answers! I'll try Versene at rt. and see how that does (keep You updated)
I also bought TrypLe(TM) Select 10X and will try that. Does anyone have any experience with this?
Have I understood that Accutase is a EDTA solution as well? Do You know which concentration? Versene is 0,53 mM.
Thank you all again!!
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