Something like a centrifuge calculator... - (Aug/13/2007 )
...does it exist?
I am electroporating cells and a lot of cells die in this protocol and they leave a lot of cell debris (membranes, dna, aso.), which may be cytotoxic and adheres to my substrate on the culture dish. This also produces background in the subsequent immunostainings.
So my idea is to do something like a cleanup centrifugation at the end of my protocol to get rid off cell debris and just lets rush through the intact cells with a higher mass. Has anyone a formula or idea to realise that?
any held appreciated
Tobi
I am electroporating cells and a lot of cells die in this protocol and they leave a lot of cell debris (membranes, dna, aso.), which may be cytotoxic and adheres to my substrate on the culture dish. This also produces background in the subsequent immunostainings.
So my idea is to do something like a cleanup centrifugation at the end of my protocol to get rid off cell debris and just lets rush through the intact cells with a higher mass. Has anyone a formula or idea to realise that?
any held appreciated
Tobi
If you centrifuge, you will pellet the cells along with alll the debris. You could try something like a density gradient centrifuge but it might take quite a bit of time and your electroporated cells might die in the mean time.
I think it is best to just electroporate and plate them as soon as possible.
To plate them as fast as possible was my first try! The cells die in the electroporation machine, so there is no chance to prevent that. Can you tell me how long such a gradient centrifugation may take? And where i can find literature?
thx for your help!
Forget gradient centrifugation, I must have been out of my mind to suggest that. In general gradient centrifugation can take 30min for some to many hours for others.
If you want to try centrifugation, try centrifuing at 100xg in for a min or 2 and then plate the cells. But if the cells are dying, I would suggest optimise the settings in a electroporating.
Do you remove the lipid debris after electroporation?
I always resuspend my cells in 10 ml of medium and suck off the white slimeball that swims on top. I suppose it's mostly membranes and maybe DNA.
If you would want to get one step further, you could spin the cells down again (15/50 ml tube, 800 rpm, 4 °C, 3-5min). This would remove anything soluble that got released from the cells and could be toxic. If you have "normal" cells (like HEK, CHO, PC12, ...) this shouldn't harm them too much.
LG
Moly