Source: Laboratory Guide to Proteins and Proteomics
Operation method
basic program
Principle
Cell fragmentation by decompression and expansion of nitrogen from a pressurized chamber is a fast and efficient homogenization method for cells and tissues, releasing intact organelles and preparing cell membranes. The principle of the method is simple: cells are placed in a pressurized chamber, and at high pressure (about 5500 kPa, equivalent to 800 psi) nitrogen is first dissolved in the cells; when the pressure suddenly disappears, nitrogen is rapidly vaporized from the solution and causes stretching and expansion of the cell membranes, which ruptures and releases the cell contents. The nitrogen compartment depressurization method is suitable for mammalian and plant cells, as well as some bacteria whose cell walls have been treated. However, this method is less efficient in lysing yeast, fungal spores, or cells with hard cell walls.
Materials and Instruments
Animal cells or tissues Move 1 Make sure the outlet is closed (but not too tight) and pre-cool the device in ice (if necessary) ② Prepare the cells or tissues to be broken. Cells in adherent culture: wash the cells gently with phosphate buffer solution (PBS) 2~3 times, and using a rubber cell scraper, scrape the cells from the culture vessel into the PBS solution. Non-adherent culture cells: centrifuge the cells at 400g for 10min, collect the cells, and wash the cells with PBS for 2~3 times. Animal tissues: filter the tissues by mechanical homogenizer, gauze net or filter sieve, and winnow the tissues. ③Collect cells by centrifugation at 480g for 5min at 4C. ④ Remove the supernatant and resuspend the cells with 10 times the volume (about 15 ml) of homogenizing solution, then transfer the supernatant to a plastic beaker (containing a magnetic stirring bar), stirring and mixing gently to obtain a homogeneous suspension. ⑤ Rotate and hold out the filter holder from the cooled cell-breaking chamber. ⑥ Add 15 ml of cell suspension to the cell crushing chamber. If the volume is large, you can use a larger model of crushing chamber, or you can use other nitrogen pressure chamber. ⑦ Reset the filter holder. ⑧ Connect the pressure chamber to the anoxic nitrogen source by tightening the screw at the top of the pressure chamber, which should already be attached to the nitrogen source and requires only a wrench. ⑨ It is best to replace the pressure chamber at a low temperature. ⑩ Allow the nitrogen to flow from the cylinder into the crush chamber and pressurize it to the desired value. A manometer must be available to show the pressure in the TK pressure chamber. The pressure required depends on the type of cell to be disrupted. The pressures generally used are: a.KB (human oral epithelial cancer cells) cells 500 psi (equivalent to 3.45 MPa) b.Rat liver cells 500psi (equivalent to 3.45MPa) c.Chicken red blood cells 1000psi (equivalent to 6.9MPa) For each experiment, step ⑩ must be chosen to best fit the expected value. In general, low pressure gives greater access to intact organelles, while high pressure results in more thorough homogenization. Cells that are difficult to lyse require multiple treatments to obtain a more thorough homogenization. The effective pressures required to break different cells are shown in Table 3.6. NOTE: Inexperienced workers handling high-pressure gases must seek expert help before using the cylinder. ⑪ Equilibrate the pressurized unit for at least 30 min to allow the nitrogen to dissolve and equilibrate in the cells. During this period stir gently 1 to 2 times with a magnetic stirrer or shake gently intermittently to ensure that the cells are in a homogeneous suspension. ⑫Place the collection vessel (e.g., beaker) next to the outlet and ice bath. ⑬ Maintain the pressure device in working condition (maintain pressure) and slowly open the outlet until the cell suspension begins to flow into the cooled collection vessel, maintaining pressure until all of the suspension has flowed out. A reasonable flow rate is 3 to 10 drops per second. When bubbles begin to flow, the device is almost empty and a faint hissing sound accompanies the flow. As the pressure is released, the cells rupture, but the micropores through the outlet also assist in cell lysis. Note: The homogenate is pushed out by high pressure and must therefore be handled with care, especially if the sample contains hazardous substances. Due to the possibility of generating gaseous lysates, the procedure must be carried out in a protected fume hood. ⑭ Before centrifuging the homogenate or subsequent operations, it must be gently stirred to dissipate the foam. ⑮ Close the outlet, but do not over-tighten it. ⑯ Turn off the nitrogen source. The effective pressure required is shown in Table 3.6. Note: Inexperienced workers handling high-pressure gases must seek expert help before using the cylinder! Caveat The nitrogen chamber decompression method is characterized as follows:① It is a gentle way to homogenize or separate cellular components because the chemical and physical pressures it applies to enzymes and subcellular components are minimal compared to other ultrasonic and mechanical homogenization methods. For example, functionally intact nuclei and mitochondria are released from most cell types.②与许多依赖压力和摩擦力的细胞裂解方法不同的是，氮减压膨胀法不会产生热，因为这种方法通过隔热膨胀来冷却样品，而不是加热它。 As a result, there is no thermal damage to proteins or organelles.(iii) By using the inert gas nitrogen, any unstable components are protected from oxidation, and nitrogen does not change the pH of the suspension medium.④ The process is fast and homogeneous because the same destructive force is applied to each cell and throughout the sample, ensuring that cell-free homogenates can be reproducibly produced.⑤ Most commercially available products are suitable for a large range of sample volumes (e.g., from about 1 ml to 1 L or more). Note: Because of the high pressure to be generated, the operation should be performed behind a protective mask. For more product details, please visit Aladdin Scientific website.
Homogenizing solution PBS
Cell Breaking Chamber Cell Scraper