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

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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

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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

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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

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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

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

Thanks

biofred

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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.

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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

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