4 replies to this topic

[Radar]

Hello everybody,

We are interested in measuring the content of an very stable metabolite in cultured macrophages. How would be the easiest way to lysate the cell pellet?

Here is my own approach:

Resuspend in distilled water and sonicate 2-4 cycles of 30 s.

As the source of the metabolite is a liposoluble drug we have added, I would prefer not to use detergents, and as the proteins are not my target, I do not need proteases inhibitors or EDTA.

Am I overlooking something? Is the use of distilled water enough to break the cells through osmotic shock?

Thank you all!

[perneseblue]

Radar, on Feb 13 2009, 08:54 AM, said:

Hello everybody,

We are interested in measuring the content of an very stable metabolite in cultured macrophages. How would be the easiest way to lysate the cell pellet?

Here is my own approach:

Resuspend in distilled water and sonicate 2-4 cycles of 30 s.

As the source of the metabolite is a liposoluble drug we have added, I would prefer not to use detergents, and as the proteins are not my target, I do not need proteases inhibitors or EDTA.

Am I overlooking something? Is the use of distilled water enough to break the cells through osmotic shock?

Thank you all!

will RNA, DNA and protein affect your assay for the drug?

[bob1]

Hypotonic solutions and a dounce or similar homogeniser:  Have a look HERE for more information on lysis methods.

Freeze/thaw cycles are pretty good too, and fairly quick if you have small volumes.

[Radar]

No, perneseblue, neither nucleic acids not proteins will interfere on the measurement, as it is through HPLC and it has a very defined signal. We can be rid of the membranes and macromolecules with centrifugation.

Thank you for the link, bob1, it is a nice and clear one. In my search I have found this one too (Millipore Cell Lysis Page). The problem we have is that our laboratory is primarily a chemistry one, and we have no Douncer or dry ice. That is why I was thinking on a buffer easy to prepare.

Does anybody know a nice page about sonicating protocols? I am looking for one at the moment and I do not find any fully satisfactory.

Thanks again!

[bob1]

Sonication depends a bit on the type of sonicator you have, bath or probe?  Sonication of cell suspensions is problematic in that it releases all the DNA and protein making the solution very viscous for quite some time until the DNA is sheared.  Sonication will also cause a lot of foaming from the solution which may not be a problem as you aren't including detergents.

My basic protocol for sonication is to tune the probe (baths aren't usually tunable) to the correct frequency for the tip, place the tip inside the solution and turn on, sonicate in 20 s bursts until desired result is achieved.  DON'T hold the tube in your hand unless you like having nerve damage, and protect your ears and wear a face mask.  Tips if they have a crack in them will sometimes spontaneously fracture under the stress of the sonication sending flying metal fragments around the room.

Dounces are cheap, try Sigma-Aldrich, we got one for about AU$100 (US$70?) they have a range between 2 and 100 ml.  Dry ice is also cheap, we can get 5kg for $20 from BOC.