I'm working with a Saccharomyces cerevisiae strain that tends to form big cell aggregates. In order to take this strain to a flow cytometer, I need to get single cells, but although I've used up to 250 mM of EDTA and strong mechanical forces, I've been unable to disaggregate the yeast's chains. Does anyone know a way to make cells separate?
İsmail Emir Akyildiz
Have you ever tried gentle, repetitive pipetting of a sample to break up weak bonds between cells? Mean trituration???
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Saif Wahid
Enzymatic treatment: Enzymes like zymolyase, lyticase, or mutanolysin can break down the cell walls and release individual cells from the aggregate. You can add these enzymes directly to the cell suspension and incubate for 15-30 minutes before washing and resuspending the cells. Be careful not to overdo the enzymatic treatment, as excessive exposure can damage or kill the cells. Sonication: This method uses sound waves to disrupt cell clusters. Briefly expose the cell suspension to ultrasonic energy using a probe sonicator or bath sonicator. This will break apart the cell aggregates into smaller clumps. Immediately after sonication, transfer the cells to a fresh tube and proceed with your desired analysis. Take note that sonication may also cause cell damage, so use caution and monitor cell viability. Mechanical disruption: Applying physical stress through vortexing, pipetting, or passage through narrow tubes can help break up cell clusters. Gently vortex the cell suspension for short periods, then immediately stop and check for cell separation under a microscope. Repeat this step several times until the desired level of disaggregation is achieved. Alternatively, pass the cell suspension through a narrow gauge needle or capillary tube to physically disrupt the cell aggregates. Chemical treatment: Some chemical agents like sodium dodecyl sulfate (SDS), Triton X-100, or saponin can solubilize lipids and break down cell membranes, leading to the release of individual cells. However, these agents can be harsh and require careful optimization to avoid cell death or altered cell properties. Start with low concentrations (e.g., 0.01-0.1%) and gradually increase them while monitoring cell viability and separation efficiency. Hypotonic shock: Cells exposed to hypotonic solutions (low osmolarity) experience water uptake, which can swell and eventually burst cell membranes, releasing individual cells. Prepare a solution with distilled water containing 10-20 mM glucose, 10-20 mM HEPES (pH 7.4), and 0.1-0.5 mM EDTA. Add this solution to the cell suspension and incubate for 10-15 minutes at room temperature. Then, rinse the cells with PBS and analyze them promptly. Flow cytometry pretreatment: If you plan to analyze the cells using flow cytometry, consider performing the above steps just before running the samples through the flow cytometer. This ensures minimal loss of cells due to sedimentation or aggregation during the analysis. Combinatorial approaches: Try combining multiple methods mentioned above for improved disaggregation efficiency. For example, first treat the cells with enzymes, followed by brief sonication, and finally, passage through a narrow tube or addition of a hypotonic solution. Yeast genetic manipulation: Consider engineering the yeast strain itself to express surface proteins that facilitate cell separation. One approach could be to introduce genes encoding for adhesion molecules with defined epitopes, allowing you to target and separate individual cells using specific antibodies.
Disaggregating yeast chains can be challenging, especially when working with certain species like Saccharomyces cerevisiae that tend to form large cell aggregates. Here are some techniques you can try to improve yeast cell separation:
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