CLaP
CLaP
CLaP is a noninvasive, laser-based labeling technique for single cells. The method uses lasers to crosslink specific cells with fluorescent tags before isolating individual cells for sequencing.
In CLaP, cells of interest are tagged by crosslinking biotin-4-fluorescein (B4F) to the cell membrane with laser irradiation. Next, streptavidin-conjugated fluorescent labels are bound to biotinylated cells. These steps can be repeated to tag multiple cell types with a variety of fluorescent tags. The tagged cells are subsequently isolated and processed to generate cDNA libraries for sequencing.
Pros:
- Noninvasive, targeted, laser-based single-cell labeling
- Enables automated, image-based cell selection
- Fluorescence-based tags can be substituted with other labels, such as electron-dense molecules
- Multicolored fluorescent stains can be used
Cons:
- Diffusion of reagents through the extracellular matrix and continuous laser illumination limit the procedure for 3-dimensional environments/tissues
- Cellular specificity may be decreased slightly in primary cell cultures.
- Image-based selection limits the potential for high-throughput applications