Hello everyone!
I am trying to use Optiprep density gradient centrifugation to get rid of cell debris from cell suspension of dissociated mouse intestinal myenteric plexus. Unlike suggested by the paper, I didn't get separation of layers after centrifugation. I changed from 2200 rpm to 1600 rpm (as the g force on the centrifuge was going beyond 500g that could harm live cells) and used MEM (2% FBS) in place of DMEM (10% FBS). Could these changes have affected the separation? The article I got this protocol is: Amber E. Kerstetter and Robert Miller, Methods Mol. Biol., 2013.
Thank you!
I've never before seen a denisity centrifugation without turning off the break and keeping acceleration as low as possible.
Maybe try this?
Thanks, Victoria Gensch for the suggestion. I tried the same protocol with minimum acceleration and no brakes during deceleration. However, it still didn't work. Could you please suggest or point me to any other Optiprep- based density centrifugation protocol to remove cell debris from a cell suspension?
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