freezing suspended cells
Posted 19 August 2009 - 03:10 AM
While freezing suspended cells in liq. N2, the general protocol says to use 20% DMSO in medium. Does anyone here know the highest limit of using DMSO for freezing suspended cells like Jurkat?
Posted 19 August 2009 - 05:05 PM
Posted 19 August 2009 - 11:44 PM
Posted 26 August 2009 - 05:18 AM
Posted 26 August 2009 - 01:50 PM
The isopropanol will freeze the cells slowly (about -1C per hour). Thaw the vial quickly, under running hot water, and put the cells into cold incomplete medium asap. After you pellet the cells, then add the warm growth medium and seed the flask.
DMSO will prevent some enzymatic reactions from taking place, and can kill the cells. If you still have problems banking cells with your current medium, try a commercially-prepared freezing medium. I use Optifreeze.
Posted 26 August 2009 - 04:38 PM
Thaw in the 37˚C waterbath and then transfer into warm medium, spin, resuspend etc. If you thaw under hot water you are liable to kill your cells, and placing them into cold medium from hot induces heatshock (and coldshock) proteins that can kill the cells.
DMSO kills the cells by damaging the membrane - it dissolves the lipids.
20% DMSO is large, I use 10% DMSO, 10% FCS and 80% medium.
Posted 26 August 2009 - 06:13 PM
After thawing the cells (under room temperature water until just melted) put the cells on ice. Transfer the cells to a 50 mL conical. Add complete media (you need the 10% FBS to help buffer the cells because the cell membranes are damaged) slowly to the cells until you have 10 mLs. Spin down gently.
DMSO is a cryopreservant because it enters the cell membranes and helps to stabilize them. It also dehydrates the cells to decrease ice crystal formation within the cells. And it decreases ice crystal formation outside of the cells.
The cell death you are seeing may be from how the cells were frozen. The cells need to be very healthy and in the log phase of growth (not overgrown) when frozen down.
Posted 28 August 2009 - 01:45 AM