An efficient method to produce clonal colonies of cancer cells using laser enabled analysis and processing (LEAP)

Many in vitro studies require a pure clonal population of cells that derive from a single cell. Traditionally this task has been performed using the inefficient manual process of ultimate limiting dilution. We have developed a novel clonal dilution technique using the Laser Enabled Analysis and Processing (LEAPTM) instrument (Cyntellect Inc. San Diego, CA). The LEAP instrument performs automated fluorescence imaging and real time image analysis to identify and measure fluorescence and morphological parameters of cells. The LEAP instrument also features a laser that can be used to manipulate targeted cells. To perform clonal dilution, cells are seeded at a low density (~10 cells/well) into each well of a 384 well plate and viably stained. The LEAP instrument will then image each well and automatically target all of the cells that are present. Then one cell will be chosen to keep (at random or based on a variety of metrics) and the others will be eliminated by laser ablation. We have successfully used this technique to produce single cell clones of HCT116 cells, a heterogeneous colorectal cancer model, in 84 percent of wells (originally containing 5 ± 2.1 cells/well). This is a marked improvement over the traditional technique of ultimate limiting dilution which produces a clone in only 33 percent of wells. The ability to efficiently produce clonal colonies has great utility in the isolation of subpopulations of cancer cells and purification of transformed cell lines.