Optimized Ki-67 staining in murine cells: a tool to determine cell proliferation

Mol Biol Rep. 2019 Aug;46(4):4631-4643. doi: 10.1007/s11033-019-04851-2. Epub 2019 May 15.


The reliable analysis of the cell cycle status has become increasingly relevant for scientific and clinical work, especially for the determination of tumor cell growth. One established method to characterize the proliferation activity of cells is the analysis of the Ki-67 protein. Ki-67 is expressed in the nucleus during the whole cell cycle except for the G0 phase. Several different protocols exist for the examination of the Ki-67 protein in tissue and cell culture, but most of them are defined for human cells. For the analysis of the Ki-67 protein in murine tissue and cell culture there is a variety of protocols existing which recommend different fixation and permeabilization reagents or special kits. In this study, we established a reliable protocol for Ki-67 staining in murine cells and tissue based on PFA fixation, which can be used not only for flow cytometry but also for immunofluorescence microscopy analysis. We tested our protocol successfully with three different Ki-67 anti-mouse antibodies in cell culture, regenerating liver tissue and mouse melanoma tumor to demonstrate the general applicability.

Keywords: Cell cycle; Cell proliferation; Flow cytometry; Hoechst staining; Immunofluorescent microscopy; Ki-67; Melanoma tumor; Mouse; Partial hepatectomy.

MeSH terms

  • Animals
  • Cell Division
  • Cell Line, Tumor
  • Cell Nucleus / metabolism
  • Cell Proliferation / physiology*
  • Flow Cytometry / methods
  • Humans
  • Ki-67 Antigen / analysis*
  • Ki-67 Antigen / metabolism
  • Mice
  • Mice, Inbred C57BL
  • Microscopy, Fluorescence / methods
  • Staining and Labeling / methods*
  • Tumor Cells, Cultured


  • Ki-67 Antigen
  • Mki67 protein, mouse