It’s an old reliable partner in protoplast preparation and extraction experiments.

Release time:

2021-11-24

Overview:

The centrifuge—a long-time companion—cannot be missing from the protoplast preparation and extraction experiment. After enzymatic digestion, the tissue homogenate is passed through a nylon mesh membrane to remove any undigested tissue fragments. The protoplasts are then washed twice using a buffer isotonic to the protoplasts themselves. Next, they are centrifuged at 50–100 g for about 1–2 minutes to remove debris. Finally, the protoplasts are resuspended in mannitol of an appropriate concentration. To reduce the proportion of debris in the protoplast suspension, you can refer to the following methods:

　　The protoplast preparation and extraction experiment cannot do without our old partner— Centrifuge It can be said to be an essential instrument for protoplast preparation and extraction experiments.

　　There are five key steps involved in protoplast preparation and extraction:

　　1. Cell fusion.

　　2. Transient expression in protoplasts: subcellular localization, protein-nucleic acid interactions.

　　3. Research on the regulatory mechanisms of ion channels and protoplasts under the influence of light, stress, and hormones using patch-clamp techniques.

　　4. An ideal material for fundamental theoretical studies on cell wall regeneration, cell division and differentiation, uptake of organelles, mechanisms of viral infection, membrane permeability, and ion transport.

　　5. It is used as an ideal material for studying calcium signaling transduction and its regulatory mechanisms in plant cells.

　　This clearly demonstrates the importance of protoplast purification. Below, we’ll provide a brief explanation using plants as an example:

　　Cellulase and pectinase can break down plant cell walls, thereby forming protoplasts. The addition of 0.6 mol/L mannitol serves to maintain the normal morphology of the protoplasts and prevent them from swelling and bursting due to water uptake. Since different plant cells have different structural characteristics, the resulting protoplasts will also vary in structure. A protoplast consists of three main components: the cell membrane, the cytoplasm, and the nucleus.

　　Pass the enzyme-digested tissue homogenate through a nylon mesh membrane to remove any undigested tissue fragments. Then, wash the protoplasts twice using a buffer isotonic to the protoplasts themselves. Centrifuge the suspension at 50–100 g for about 1–2 minutes to pellet and remove debris. Finally, resuspend the protoplasts in mannitol of an appropriate concentration. The following methods can be referred to reduce the proportion of debris in the protoplast suspension:

　　1. Optimize the samples in advance—for example, peel off the outer skin (but only for samples where the research subject does not include the outer skin portion), and discard tissues that are hard in texture or damaged.

　　2. Reduce the centrifuge’s rotation speed and duration to remove debris remaining in the supernatant.

　　3. If the initial amount of protoplasts is sufficiently large, consider using flow cytometry sorting to remove debris.

　　The above is an overview of the application of centrifuges in protoplast preparation and extraction experiments. If you’d like to learn more about centrifuges, please stay tuned for updates from Shanghai Luxiangyi Centrifuge Instrument Co., Ltd.

Using crossmatching as an example, let’s explore the importance of centrifuges in medical laboratories.

Take the laboratory centrifuge as an example to analyze rotational speed calibration.