Ki-67 Cell Proliferation Assay

A quantitative method to assess cell proliferation is an essential prerequisite for drug screening in vitro. Studying cell proliferation in primary isolated cells or permanent cell lines provides an important parameter for improving the cytocompatibility of candidates, particularly in the context of cytotoxicity testing.

The Ki-67 protein is a nuclear antigen associated with cell proliferation and can be used as a marker for cell proliferation assay because it is expressed throughout the active cell cycle (G1, S, G2, and M phases) except for the resting phase (G0). The main advantages of assaying Ki-67 protein are that various techniques can be used: formalin-fixed paraffin specimens and frozen tissue samples by microscopy, single cell suspensions by flow cytometry, and cell lysates by western blot.

The following is a detailed protocol for the detection of Ki-67 protein by immunohistochemistry.

Materials and Equipment

The required materials and equipment are shown in the table below:

Slide Preparation

• Coat coverslips with polyethylineimine or poly-L-lysine for 1 hour at room temperature.
• Rinse coverslips with sterile H₂O (three times, 1 hour each time).
• Allow coverslips to dry completely and sterilize under UV light for at least 4 hours.
• Culture cells on the glass coverslips or prepare cell smears.
• Rinse briefly with PBS.
  Note: For wash buffers, we recommend 1X PBS containing 0.1% Tween 20.

Fixation

The cells can be fixed by one of the following methods:

• Incubating the cells in 100% methanol (chilled at -20°C) for 5 min at room temperature.
• Using 4% paraformaldehyde in PBS (pH 7.4) for 10 min at room temperature.
• Wash three times with ice cold PBS.

Permeabilization

• For intracellular proteins, it is very important to permeabilize the cells. Methanol fixed samples do not require permeabilization.
• Incubate the samples for 10 min with PBS containing 0.1-0.25% Triton X-100.
  Note: Triton X-100 is the most commonly used detergent to increase the penetration of the antibody. However, it is not appropriate for membrane-associated antigens because it destroys the cell membranes.
• The optimal percentage of Triton X-100 should be determined for different proteins.
• Wash cells for 5 min with PBS.

Antigen Retrieval (Optional)

Check the product information for advices on each primary antibody being used.

• Preheat the antigen retrieval buffer to 95°C.
• Place the coverslips carefully in the antigen retrieval buffer and make note of which side of the coverslips the cells are on.
• Heat the coverslips at 95°C for 10 min.
• Remove the coverslips from the antigen retrieval buffer and immerse them in PBS with the side containing the cells facing up.
• Wash cells for 5 min with PBS.

Blocking and Immune-staining

• Incubate cells with 5% goat serum for 30 min to block unspecific binding of the antibodies (see the antibody datasheet for recommendations).
• Incubate cells with the diluted primary antibody in a humidified chamber for 1 hour at room temperature or overnight at 4°C.
• Wash the cells three times in PBS and 5 min each time.
• Incubate with the secondary antibody for 1 hour at room temperature in the dark.
• Decant the secondary antibody solution and wash three times with PBS for 5 min each.

Counter Staining

• Incubate the cells with 0.1-1 μg/mL Hoechst or DAPI (DNA stain) for 1 min.
• Rinse with PBS.

Mounting and Image

• Mount the coverslip with mounting medium to prevent drying.
• Image and analyze the stained cells under the microscope.

References

1. Romar G. A. et al.; Research techniques made simple: techniques to assess cell proliferation. J Invest Dermatol, 2016, 136 (1): 1-7.
2. Klein C. L. et al.; A new quantitative test method for cell proliferation based on detection of the Ki-67 protein. J Mater Sci Mater Med, 2000, 11(2):125-132.

For research use only. Not for any other purpose.