look for the RIPA recipe and use the same. Freeze-thaw is not suggested use physical disruption techniques with RIPA treatment instead...

If you can't find a specific protocol detailing the buffer to use for what you want to do, I would start with a simple buffer such as 25 mM HEPES, 10 mM NaCl or KCl and 1 mM EDTA at pH 7.4.

Freeze thaw lysis certainly works at lysing cells, but it takes time. You freeze your sample tube on dry ice or in liquid N2 then rapidly thaw in warm water. You'll need to do this 3-5x times or so though to get maximal lysis, which will take a while.

If you are aiming to extract membrane vesicles, then you will need to avoid detergents for lysis and use physical methods. RIPA buffer will certainly be too aggressive because of the denaturing ionic detergents that it contains. What sort of volume of cells are we talking about here? If you are only lysing a small amount (less than 2 ml or so), then passing them through a 27 g needle 10-20x on ice works very effectively. You'll probably need to break up clumps first with a 21 g or similar needle. For larger volumes (5-50 ml), a Dounce or Potter homogeniser is very effective. Beyond 50 mL you'd be best looking at a pressure cell disruptor or French press etc. Of course, this all depends on what you have access to.

Hello Sanggeeta,

The most popular method to extract cancer cell membrane vesicles involves osmosis-based cell lysis with a mild hypotonic solution, followed by mechanical membrane disruption with a homogenizer. Discontinuous gradient centrifugation removes intracellular biomacromolecules, intracellular vesicles, and nuclei, and the membrane-rich fraction is washed with isotonic buffers to obtain membrane vesicles. Cancer cells will require milder lysis conditions and greater ultracentrifugation speeds compared to non-nucleated cells.

A typical protocol for cell membrane vesicle preparation includes three basic steps.

1. First, the parental cells are broken down by lysing with a hypotonic buffer. A cell placed in a hypotonic solution swell and expands until it finally bursts by a process known as cytolysis. A hypotonic buffer is therefore used to lyse the cells and release the cytoplasmic fraction.

2. Second, the mixture of cell membranes and other cellular components, such as cell nucleus and cytoplasmic organelles, are separated by centrifugation. The centrifugation method may differ depending on the cell type. For instance, preparation of eukaryote cell membranes requires discontinuous sucrose gradient centrifugation to separate the membrane from other cell components and nuclei, whereas this gradient centrifugation step is dispensable for preparation of membranes from nucleus-free cells, such as RBCs.

3. Third, the collected cell membrane is physically broken to yield cell membrane vesicles of the size of interest.

You may suspend the cells in a hypotonic lysing buffer consisting of 20 mM Tris-HCl (pH = 7.5), 10 mM KCl, 2 mM MgCl2, and 1% phenylmethanesulfonyl fluoride (PMSF) and lyse in ice bath for 15 min. 

You may also try hypotonic solution containing 10mM HEPES(pH 7.9), 10mM KCL, 0.1mM EDTA and 0.1mM EGTA. Just before use, add protease and phosphatase inhibitor cocktail to a final 1× concentration and DTT to a final concentration of 0.5 mM.

Yes, you may use the freeze-thaw lysis method. After treating the cells with hypotonic lysis buffer, the entire solution may be freeze and thawed repeatedly, following centrifugation at 1000 × g for 10 min at 4 °C. The supernatant may be centrifuged at 14,000 × g for 30 min at 4 °C to obtain the membrane fragments. The final fragments may be collected in PBS for subsequent analysis.

Best.

Thank you for your kind feedback @ İsmail Emir Akyildiz and Samuel Davis