Single Cell Sampling Using Laser Capture Microdissection: A Technique to Harvest Target Cells from a Heterogeneous Cell Population

JoVE Encyclopedia of Experiments

Cancer Research

0 weergaven • 2:30 min • April 30th, 2023

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Laser capture microdissection helps to procure a single target cell from a cell mixture prepared on a glass slide. Begin by taking a suitable glass slide coated with a UV-permeable, biochemically inert membrane on its surface. Expose the slide to UV light to render it more hydrophilic for better cell adherence.

Assemble the slide into a cytocentrifuge apparatus.  Add cell suspension onto the funnel port of the apparatus. Cytocentrifuge to allow the centrifugal force to concentrate and deposit a monolayer of cells on a small area on the glass slide. Allow the slide to air-dry.

Place the slide, sample side up, under a laser microdissection microscope. Define the region surrounding the cell of interest. Now, focus the laser beam to dissect the target-cell-containing region along with the membrane coating. Next, defocus the laser beam and pulse it to generate pressure. This energy catapults the microdissected region into the inverted cap of a pre-assembled collection tube containing a buffer placed above the slide.

Retrieve the tube and close its cap. Spin the tube to bring the buffer containing the single target cell to the tube bottom. Refrigerate the tube until further analysis.

For laser mircodissection, cytocentrifuge the entire 300 microliters of cell suspension onto a membrane-coated slide at 300 x g for 5 minutes. Then, place the membrane-coated slide on a microscope capable of laser mircodissection. Next, pipet 4.5 microliters of cell lysis master mix into the cap of the 0.2-milliliter PCR tube.

Place the cap above the sample and harvest a single cell by laser microdissection. Directly thereafter, check for isolated cells in the PCR cap. Remove the PCR tube from the laser mircodissection microscope and close the tube. Collect all the liquid at the bottom of the tube with a short spin.

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Laatst bijgewerkt: 22 augustus 2026