In vivo DNA replication labeling
DNA replication in higher eukaryotes takes place in a well-defined spatial and temporal manner. A transient permeabilization that allows the uptake of macromolecules from the surrounding medium can be achieved by a number of methods. More suitable regarding loading efficiency and viability are methods creating slight mechanical damage to the cell membrane in the presence of nucleotides. This can be achieved by shaking a labeling solution with small glass beads over a cell layer or by detaching a cell layer with a cell scraper. The best results in terms of labeling efficiency, reproducibility, and amount of nucleotides needed were obtained by a modified scratch-loading protocol, termed as scratch replication labeling. This protocol can be applied to any adherently growing cell line. It allows the labeling of a high number of cells within one experiment with little effort. The fraction of replication-labeled cells depends on the density of scratches applied and number of cells that are in S phase.