Laser Capture Microdissection (LCM) is a proven technique for isolating pure cell populations for downstream molecular analysis. Combined UV laser cutting and LCM using the IR laser, found only with the Veritas™ Microdissection System, allows for rapid and precise isolation of larger numbers of cells, while maintaining cellular and nucleic acid integrity necessary for downstream analysis. We have shown that these established techniques can also be used for isolating living cells, providing opportunity for a wide range of research applications.
To validate the protocol, cells were seeded onto a clean frame PEN membrane slide and allowed to grow. When confluent, the media was removed and fresh media was added to the chamber. A clean cover glass was placed over the chamber, creating a mini cell culture environment, enabling extended cell survival during microdissection.
The slide was transported to the Veritas, cleaned and loaded onto the system with the cover glass facing down (Figure 1). While settings were optimized for cell lines used in the study (TM3 and SKBR3), general guidelines were established for overall live cell microdissection success. It is recommended to always use CapSure® Macro LCM caps for live cell microdissection. Further, the user should perform microdissection at 10X or 20X objective and activate the IR capture laser first, before the UV cutting laser. For optimal visualization of the live cells, the Visualizer should be turned off when beginning the session. Specific laser and cut and capture settings will vary depending on cell line and preparation and should be optimized as needed.
Following microdissection, the cap was moved to the QC Station to visualize the cells. After confirming the presence of desired cells, the cap was offloaded, removed from the system and placed face up in a clean Petri dish. The microdissected cells were first rinsed, while still on the cap, and then Trypsin-EDTA was pipetted onto the cap and incubated at room temperature for 5 minutes. After incubation, the Trypsin-EDTA was pipetted up and down several times to ensure a single cell suspension, then transferred into a desired growth vessel containing fresh media. The cells were then placed into an incubator and allowed to reculture.
During protocol validation, some caps containing live cells were placed into PicoPure® extraction buffer for RNA isolation. Isolated total RNA was quantitated and the quality was assessed using the Agilent Bioanalyzer (Figure 2). The quantity and quality of RNA achieved after microdissection proved high enough for direct downstream processing, such as RNA amplification for use in molecular analysis.
Using this robust technique, possible only with the Veritas Microdissection System, microdissected live cells remain viable for subsequent reculture or for direct processing for use in downstream applications. For the complete protocol, please visit Web site (external).
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