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Cell lysis and osmotic shock - (Feb/21/2007 )

Hello,

Is anyone know a protocol to lyse cell by osmotic shock or by Freeze and thaw???
Thanks a lot

biofred

-Biofred-

QUOTE (Biofred @ Feb 21 2007, 09:41 AM)
Hello,

Is anyone know a protocol to lyse cell by osmotic shock or by Freeze and thaw???
Thanks a lot

biofred


many cell lysis buffers are hypoosmolar for cells; freezing and thawing may not be useful as lysed lysosomes do harm by releasing proteases before protease inhibitors get contact to proteases

-The Bearer-

Hi bearer thanks for your answer, the content of the cell is not important for me. I just want to recover my bacteria inside the cell. Maybe freezing the cell is also not good for bacteria. Do you have a simple recipe for lysis buffer without detergent?

Thanks

-Biofred-

QUOTE (Biofred @ Feb 21 2007, 11:58 AM)
Hi bearer thanks for your answer, the content of the cell is not important for me. I just want to recover my bacteria inside the cell. Maybe freezing the cell is also not good for bacteria. Do you have a simple recipe for lysis buffer without detergent?

Thanks


lysis buffers contain detergents to disrupt membranes; so a lysis buffer w/o detergents may not virtually a work to lyse cells; you mean a homogeneization buffer which work w/o detergents if you physically disrupt your cells or by sonifying or freeze/thawing

-The Bearer-

QUOTE (Biofred @ Feb 21 2007, 03:58 AM)
Hi bearer thanks for your answer, the content of the cell is not important for me. I just want to recover my bacteria inside the cell. Maybe freezing the cell is also not good for bacteria. Do you have a simple recipe for lysis buffer without detergent?

Thanks


We used to do an assay to determine salmonella numbers, both internal and external numeration. Our cells were grown in 6 well plates and infected with different MOI of Salmonella. To determine intracellular numbers, the cells were lysed simply with distilled water

-Rhombus-

It's looks interessant for me. How much time do leave the cell with distilled water?

Thanks

biofred

-Biofred-

QUOTE (Biofred @ Feb 22 2007, 12:09 AM)
It's looks interessant for me. How much time do leave the cell with distilled water?

Thanks

biofred


Dear Biofred,

To determine extracellular numbers just take off media from multi well plate

To determine intracellular numbers add sterile distilled water. For our cells, J774 (Murine macrophages), 3 minutes was used. We observed our cells under the microscope and could see them bursting.

You need to optimise the time FOR YOUR PARTICULAR CELL.

-Rhombus-

Thank you very much I will try this afternoon.

I use Mf4/4 (murine macrophage) and J774 like you.

I hope it will work also for me!

Have a nice day

biofred

-Biofred-