Cell Proliferation Assay2022-11-09T14:49:21-05:00


  • The Ki-67 protein is a cellular marker for proliferation (1).  During interphase, the Ki-67 antigen can be exclusively detected within the cell nucleus, whereas in mitosis most of the protein is relocated to the surface of the chromosomes (2). Ki-67 protein is present during all active phases of the cell cycle (G1, S, G2, and mitosis), but is absent in resting (quiescent) cells (G0) (3). Cellular content of Ki-67 protein markedly increases during cell progression through S phase of the cell cycle (4).  Ki67 index is frequently cited by pathologists when diagnosing degree and severity of cancer patients to identify those who derive greater benefit from chemotherapy (5).
  • Ki-67 is an excellent marker to determine the growth fraction of a given cell population. The fraction of Ki-67-positive tumor cells is often correlated with the clinical course of cancer. The prognostic value of Ki67 for survival and tumor recurrence have repeatedly been proven.
  • In the screening of antiproliferative drugs for cancer, Ki67 is a commonly used marker to determine the fraction of cells currently in a proliferative state. Antiproliferative drugs such as cisplatin and paclitaxel markedly decrease the fraction of proliferating cells as measured by Ki67.
  • 3D cell culture models more closely recapitulate the in vivo tumor environment, and are an effective tool for the evaluation of antiproliferative effects of promising anti-cancer drug compounds.
  • Using High Content Screening with 3D cell models offers a medium-throughput approach to screening the antiproliferative activity of test compounds.


InstrumentMolecular Devices ImageExpress Micro Confocal
Analysis MethodHigh content screening
MarkersImmunolabeling for Ki67+ cells
DAPI (total cell count)
Cell Types AvailableHepG2, A549, BT549, MCF-7, other ATCC cell lines available on request
Test Article Concentration8 point assay (0.05, 0.1, 0.5, 1, 5, 10, 50, 100 µM)
(custom concentrations available)
Number of Replicates3 replicates per concentration
Quality Controls0.5% DMSO (vehicle control)
Paclitaxel (positive control)
Cisplatin (positive control)
Test Article Requirements50 uL of 20 mM solution or equivalent amount of solid
Data DeliveryDose response curves, IC50 values, total cell counts, Ki67% for each test concentration

General Procedure

  1. Tumor spheroids grown by seeding 1000 cells/well into ULA U-bottom plates
  2. Tumor spheroids grown to approximately 200 μm in diameter
  3. Treatment with test compounds
  4. After 24 hours, tumor spheroids are fixed and processed for immunolabeling
  5. Tumor spheroids are labeled with DAPI and anti-Ki67 antibody
  6. Tissue clearing is applied to render tumor spheroids transparent
  7. High content imaging is conducted on well plates
  8. Images are analyzed to quantify total number of cells and total Ki67+ cells


Figure 1. Images of single slice of Z-stack obtained from High Content Imaging of tumor spheroids treated with cisplatin. Data is quantified by automated image analysis to count total number of cells, and number of Ki67+ cells

Figure 2. Representative data showing the observed Ki67% of NCI-H2170 tumor spheroids treated with various concentrations of cisplatin. The IC50 value was found to be 11.4 μM.

Spheroid Spatial Dose Response Graph

Figure 3. Representative data depicting a spatial dose response curve. With tissue clearing, the effect of cisplatin on cell proliferation as measured by Ki67% can broken down into various cell populations, here comparing the dose dependent response by position of the cells within the tumor spheroid. Dose response can be measured and compared between the outermost third of cells (blue) against the cells within the interior of the tumor spheroid (green, middle layer, and red, the innermost core of cells). This can be important when judging the effect of drugs that are meant to target the innermost hypoxic cells (e.g. Tirapazamine).


  1. Scholzen T, Gerdes J (March 2000). “The Ki-67 protein: from the known and the unknown”. Journal of Cellular Physiology182 (3): 311–22.
  2. Cuylen S, Blaukopf C, Politi AZ, Müller-Reichert T, Neumann B, Poser I, Ellenberg J, Hyman AA, Gerlich DW (July 2016). “Ki-67 acts as a biological surfactant to disperse mitotic chromosomes”Nature535 (7611): 308–12.
  3. Bruno S, Darzynkiewicz Z (January 1992). “Cell cycle dependent expression and stability of the nuclear protein detected by Ki-67 antibody in HL-60 cells”. Cell Proliferation25 (1): 31–40.
  4. Darzynkiewicz Z, Zhao H, Zhang S, Lee MY, Lee EY, Zhang Z (May 2015). “Initiation and termination of DNA replication during S phase in relation to cyclins D1, E and A, p21WAF1, Cdt1 and the p12 subunit of DNA polymerase δ revealed in individual cells by cytometry”Oncotarget6 (14): 11735–50.
  5. Sonnenblick A, Francis PA, Azim HA, de Azambuja E, Nordenskjöld B, Gutiérez J, Quinaux E, Mastropasqua MG, Ameye L, Anderson M, Lluch A, Gnant M, Goldhirsch A, Di Leo A, Barnadas A, Cortes-Funes H, Piccart M, Crown J (2015). “Final 10-year results of the Breast International Group 2-98 phase III trial and the role of Ki67 in predicting benefit of adjuvant docetaxel in patients with oestrogen receptor positive breast cancer”. European Journal of Cancer (Oxford, England : 1990)51 (12): 1481–9.

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