Bromodeoxyuridine immunofluoresence and differential interference contrast imaging combination can precisely segregate adherent monolayer cells into specific cell-cycle phases

Abstract

Most cellular-level cancer studies involve the identification of the cell cycle phases in which individual cells are progressing through. Traditional methods such as Fluorescent Activated Cell Sorting (FACS) require several treatments before harvesting the cells—procedures which alter cellular architecture. This study describes a novel method of the cell cycle analysis that preserves the cellular morphology and architecture with minimal in situ milieu perturbation. Primary rat skin fibroblasts were isolated and cultured at standard conditions. The cells were stained with anti-BrdU and examined with LSM 510 laser scanning microscopy. S-phase cells incorporated BrdU while M-phase appeared smaller and spherical. Damaged cells also tended to round-off in shape but, unlike M-phase cells, they did not bind anti-Phospho H3 antibody. G1 and G2 phases did not incorporate BrdU or Phospho H3. The two gap phases were differentiated on the basis of their sizes and subtleties in their shapes. The method is technically simple and less time-consuming while preserving the cellular in situ architecture. Due to its simplicity and accuracy the technique can be easily employed in resource-limited laboratories. Further studies are needed to verify the usefulness of the technique in clinical diagnostics such as cancer biomarkers.