Name: lysis methods for preparing tissue samples
Uploaded: 2023-04-30T12:46:58.000Z
Duration: 1 min 32 s
Channel: JoVE

Tissue homogenization techniques disrupt tissue architecture and lyse cells to isolate subcellular organelles or macromolecules, such as nucleic acids and proteins.
Depending on the sample and the intended downstream application, homogenization can be performed using mechanical or non-mechanical methods.
Solid-based mechanical methods such as a tissue grinder or bead beater are used for&#160; hard or fibrous tissue samples like bone and muscle.&#160;
While a tissue grinder uses a manually or automatically operated mortar and pestle to grind a sample, the bead beater uses collision between the sample and the beads to break apart the tissue.
Ultrasonication is a liquid-based mechanical homogenization method that can shear cells in suspension using high-frequency sound waves generated by a vibrating probe.&#160;
Non-mechanical techniques include enzymatic, biological, or chemical homogenization methods.
These comprise the use of enzyme cocktails containing collagenase or trypsin or chemical agents like detergents or chaotropes, freeze-thaw cycles, or osmosis to breach the cell wall and plasma membrane and release the cell&#39;s contents into the solution.
Non - mechanical methods are often combined with

tissue homogenization and cell lysis

Tissue Homogenization and Cell Lysis

Tissue homogenization involves disintegrating tissue architecture and lysing cells, and is an early step in isolating and analyzing cellular components. The method used for homogenization depends on the sample type, the amount of sample available, the analyte to be obtained, and the sensitivity of the method. These methods are broadly classified as mechanical and non-mechanical methods.
Mechanical methods of tissue homogenization
These methods rely on applying external physical force to disrupt tissues and cells. They make use of specialized tools and instruments for homogenization. These instruments use grinding, shearing, blending, beating, or shock to disintegrate the sample. For example, in a French press, the sample is pushed through a small opening under pressure which causes the cells to disrupt. Other homogenizers such as Waring blenders and rotor-stators cut and shear the tissues into significantly smaller sizes.
Non-mechanical methods of tissue homogenization
Non-mechanical lysis methods involve chemical disruptions rather than physical forces to lyse the cells. The tissue is homogenized in a lysis buffer that regulates pH, ionic strength, osmotic strength, and enzymatic activity. The lysis buffer thus aids cell lysis and protects the cell components from damage.
While the enzymes of the lysis buffer help degrade the extracellular matrix of tissues to release individual cells, the surfactant or detergent helps disrupt cell membranes and denature proteins. Sodium dodecyl sulfate (SDS) and Triton-X 100 are two popularly used detergents in these buffers. Another component, the chaotropes, disrupt weak interactions between molecules, thus denaturing the proteins and keeping nucleic acids intact during isolation.
Other non-mechanical physical methods involve using temperature cycles in which the sample is frozen on dry ice or in an ethanol bath and then thawed at room temperature or 37&#8451;. These repeated cycles cause the cell membranes to weaken and rupture. Cell membranes can also be ruptured by osmotic imbalance by placing them in a hypotonic or hypertonic solution. The inward or outward movement of water due to the osmotic gradient causes the cells to swell and burst, or shrink and collapse, releasing their internal contents.
While numerous methods and tools are available for homogenization, each has pros and cons that must be evaluated based on the specific requirements. Often, mechanical methods alone may not wholly or efficiently homogenize a sample. In such cases, mechanical methods may be combined with non-mechanical methods for complete homogenization.
While numerous methods and tools are available for homogenization, each has pros and cons that must be evaluated based on the specific requirements. Often, mechanical methods alone may not wholly or efficiently homogenize a sample. In such cases, mechanical methods may be combined with non-mechanical methods for complete homogenization.

Tissue homogenization involves disintegrating tissue architecture and lysing cells, and is an early step in isolating and analyzing cellular components. ...

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Cell Biology

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Analyzing Cells and Proteins

KEY TERMS AND CONCEPTS

lysis methods for preparing tissue samples

Tissue sample preparation often starts with homogenization and cell lysis. This step breaks tissue structure and opens cells so their parts can be studied. The best method depends on the sample type, the amount of sample, the analyte to be collected, and how sensitive the method is.

Tissue homogenization methods are usually grouped into mechanical and non-mechanical methods. Mechanical methods use external physical force to disrupt tissues and cells. These methods may use grinding, shearing, blending, beating, or shock to break the sample into smaller pieces.

Some mechanical tools are specialized for this job. In a French press, the sample is forced through a small opening under pressure, which disrupts the cells. Waring blenders and rotor-stators also cut and shear tissue into much smaller fragments.

Non-mechanical lysis methods use chemical disruption instead of physical force. The tissue is placed in a lysis buffer, which helps control pH, ionic strength, osmotic strength, and enzymatic activity. This buffer supports cell lysis and helps protect cell components from damage.

The enzymes in the lysis buffer can break down the extracellular matrix, which helps release individual cells. Surfactants, also called detergents, disrupt cell membranes and denature proteins. SDS and Triton-X 100 are two commonly used detergents in these buffers. Chaotropes are another buffer component, and they disrupt weak interactions between molecules. This helps denature proteins while keeping nucleic acids intact during isolation.

Some non-mechanical methods use physical conditions instead of chemicals. One method uses repeated freeze-thaw cycles. The sample is frozen on dry ice or in an ethanol bath and then thawed at room temperature or 37°C. These cycles weaken cell membranes until they rupture.

Cells can also burst from osmotic imbalance. In a hypotonic or hypertonic solution, water moves in or out because of the osmotic gradient. The cells may swell and burst, or shrink and collapse, which releases their internal contents.

Each homogenization method has advantages and limitations. In many cases, mechanical methods alone do not fully or efficiently homogenize a sample. For that reason, mechanical and non-mechanical methods are often combined to achieve complete homogenization.

Lysis Methods for Preparing Tissue Samples

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Tissue sample preparation often starts with homogenization and cell lysis. This step breaks tissue structure and opens cells so their parts can be studied. The best method depends on the sample type, the amount of sample, the analyte to be collected, and how sensitive the method is. Tissue homogenization methods are usually grouped into mechanical and non-mechanical methods. Mechanical methods use external physical force to disrupt tissues and cells. These methods may use grinding, shearing,...

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key terms and concepts

Learn how tissue samples are prepared by mechanical and non-mechanical lysis methods, including blenders, buffers, detergents, freeze-thaw cycles, and osmotic imbalance.

Lysis Methods for Preparing Tissue Samples | JoVE K12

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Lysis Methods for Preparing Tissue Samples

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