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infection assay-buffer that lyses mammalian cells but not bacteria

cell biology

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#1 micronagu

micronagu

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Posted 26 June 2013 - 06:46 PM

Dear Experts,

this is nagu, doing my PhD on Helicobacter pylori. Here is my question,

I am infecting the AGS mammalian cell line with Helicobacater pylori and after prescribed incubation time i have to lyse the mammalian cell line for western blot analysis. Currently i am using RIPA buffer with NP 40 and i have a thought that it may lyse the attached bacteria on the mammalian cell surface. I am asking here your suggestions what is the lysis buffer is better to lyse the mammalian cells not the attached bacteria. I refered some from published articles. but I need your valuable suggestions and experience.

thanks in advance.

nagu

#2 rhombus

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Posted 28 June 2013 - 05:18 AM

Dear Microagu,

We have used distilled water to release the Salmonella bacteria which become internalised within murine macrophage cell line (J774).

We first washed the cell monolayer x 3 with PBS to remove extracellular bacteria. We were interested in using different multiplicity of infection (MOI) i.e 1, 10, 100 and 1,000. We were then growing the bacteria AND not the cells to assess the numbers of internalised bacteria.

If I am understanding your protocol you could:-

Wash your cell monolayer with PBS possibily as many as 3 times. This should remove the Helicobacter

BUT you need to check that this is the case. Also you need to check that the Helicobacter is not INTERNALISED. You could do this by Confocal microscopy/3D imaging.

If they are internalised you have a problem i.e. you will have to use Triton or RIPA to lyse the mammalian cells.....THEN use the confocal to see if the cells are lysed and the bacteria ARE NOT. Repeat different concentrations of lysis buffer and time of incubation IF both are lysed

Once this has been checked then you should you a lysis buffer containing protease inhibitors i.e. to reduce proteolytic attack of cellular proteins by proteases.

I do not know if this is what you were after

Kindest regards

Uncle Rhombus





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