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Cell lysis - (Feb/16/2007 )

Hello guys,

I have a simple question for you. I need to lysate some cells ( Murine macrophage, Mf4/4: 60.000Cell/well) in 24well. The medium is RPMI. Normally I lysate my cell with 200µl of PBS1X-Triton 0,1%. But for these experiment I can't remove the medium of each well. Do you know if the result will be same if I add 20µl of PBS1X-Triton 1%?? Or do you have an other solution to lysate my cell??

Thanka a lot for your help!!

biofred

-Biofred-

QUOTE (Biofred @ Feb 16 2007, 11:36 AM)
Hello guys,

I have a simple question for you. I need to lysate some cells ( Murine macrophage, Mf4/4: 60.000Cell/well) in 24well. The medium is RPMI. Normally I lysate my cell with 200µl of PBS1X-Triton 0,1%. But for these experiment I can't remove the medium of each well. Do you know if the result will be same if I add 20µl of PBS1X-Triton 1%?? Or do you have an other solution to lysate my cell??

Thanka a lot for your help!!

biofred


if RPMI is fed with FCS you will have much serum proteins predominantly albumine in your lysate, and all other ingredients of medium; this may disturb sequential analysis of lysates;

an alternative to chemical lysis is sonification

-The Bearer-

QUOTE (Biofred @ Feb 16 2007, 11:36 AM)
Hello guys,

I have a simple question for you. I need to lysate some cells ( Murine macrophage, Mf4/4: 60.000Cell/well) in 24well. The medium is RPMI. Normally I lysate my cell with 200µl of PBS1X-Triton 0,1%. But for these experiment I can't remove the medium of each well. Do you know if the result will be same if I add 20µl of PBS1X-Triton 1%?? Or do you have an other solution to lysate my cell??

Thanka a lot for your help!!

biofred


Hi
why don't you can remove the medium of each well? If not, I'm workin with monocytes cells and to lyse them I use a buffer containing triton, like you!
But in my laboratory exist many other protocol of lysis... therefor, if you want specify me a little your aim

-boumbo_doc-

Hello boumbo_doc,

In fact my cells are infected by BCG and I want to see the evolution of my bacteria in time. I can't remove the media because the cell dye and some bacteria are released outside. So in time I lyse my cell and after mesure the luciferase activity of the lysate (strain transform with plasmid who express luciferase activity). It's why I need a solution to lysate the cell without removing the media??

What do you think?

biofred

-Biofred-

QUOTE (Biofred @ Feb 19 2007, 08:43 AM)
Hello boumbo_doc,

In fact my cells are infected by BCG and I want to see the evolution of my bacteria in time. I can't remove the media because the cell dye and some bacteria are released outside. So in time I lyse my cell and after mesure the luciferase activity of the lysate (strain transform with plasmid who express luciferase activity). It's why I need a solution to lysate the cell without removing the media??

What do you think?

biofred


Hi,

well, what do you think about sonication? is true that your case it's interesting but do you culture your cells in 6, 12... wells?

-boumbo_doc-

Hi, in fact my cells are in 24 or 96 wells so it's a lot of samples to sonicate, is not?

biofred

-Biofred-