Description
The expression of the proliferation related nuclear antigen Ki-67, also known as Ki67 or MKI67, is a cellular marker for proliferation which is only detected in dividing cells. Ki-67 is a non-histone protein characterized as a nuclear matrix-associated proliferation-related antigen present throughout the cell cycle, but not in resting, quiescent, cells (G0 phase). Ki-67 is widely used in routine pathology as a proliferation marker to measure the growth fraction of cells in human tumors. The Ki-67 protein present in the nucleus of proliferating cells is released upon cell lysis, and the Ki-67 protein extracted in the lysate is quantified using our Ki-67 HTRF assay. The Ki-67 quantitation assay is an alternative to BrdU or EdU incorporation assays, which are exogenous proliferation markers incorporated into the newly synthesized DNA of dividing cells. The scope of Ki-67 is similar to that of the proliferating cell nuclear antigen (PCNA).
Assay principle
The Ki-67 quantification kit is based on a sandwich format involving two specific antibodies, respectively labelled with Cryptate (donor) and d2 (acceptor). The Ki-67 protein contained in the proliferating cell nucleus and extracted in cell lysates binds to monoclonal anti-Ki67 Eu Cryptate and monoclonal anti-Ki67 d2 conjugates. When the dyes are in close proximity, the excitation of the donor with a light source (laser or flash lamp) triggers a Fluorescence Resonance Energy Transfer (FRET) towards the acceptor, which in turn fluoresces at a specific wavelength (665 nm). The specific signal modulates positively in proportion to Ki-67 concentration.
Assay protocol
The assay protocol, using a 384-well small volume white plate or a Cisbio low volume 96-well plate (20 µL final), is described on the right. 4 µL of sample (lysate) + 12 µL of lysis buffer, or 16 µL of standard are dispensed directly into the plate for detection by HTRF reagents. The antibodies labelled with HTRF donor and acceptor can be pre-mixed and added in a single dispensing step to further streamline the assay procedure. The assay can be run in 96- to 384-well plates by simply resizing each addition volume proportionally.
Ki-67 protein expression on starved cultured cells upon stimulation/re-feeding with 24h 10% FCS
A serum starvation and refeeding process was used to imitate the cell cycle using human epithelial A549 cell line derived from a lung carcinoma tissue. First, Ham's F-12 medium without FCS (supplemented with 0.5% BSA, culture grade) was used to incubate A549 cells for 72 h to synchronize them (60K or 120K cells per well in 100µL medium.). The medium was then changed to complete medium in six wells (10% FCS), keeping six wells for starved control.On the first microplate, the medium was removed. Cells were then lysed in 50 µL 1X Lysis buffer and 4 µL lysate were transfered to a 384 well white microplate. 12 µL 1X lysis buffer were added, following the Ki-67 HTRF protocol.The same experiment was run in parallel on the second microplate to compare with a brdU incorporation assay, using a commercial BrdU-ELISA kit. Briefly, BrdU was added (10µL/well) after 2h incubation. Medium was removed and cells were fixed and after blocking (1% BSA 30 min). An anti-BrdU peroxidase conjugate was added (90 min incubation RT), followed by the washing and coloration steps.Ki-67 expression increased in cell lysate upon stimulation with FCS compared to the starved cell control. In parallel, BrdU incorporation increased in FCS stimulated cells. The Ki-67 expression correlates with the BrdU incorporation assay. It should be noted that the assay window was larger in the Ki-67 HTRF assay compared to BrdU assay.
Differential Ki-67 protein expression in confluent and non-confluent A549 tumor cells
A549 cells were cultured in two T175 flasks in Ham's F-12 complete medium (10% FCS), using two seeding concentrations to obtain either confluent cells or non-confluent cells. The adherent cells were washed with PBS, then dissociated (5 mL cell dissociation buffer), scraped with a rubber policeman and transferred to three 1.5 mL microtubes (10.E6 cells/tube) as technical replicates. After centrifugation (5 min, 400 RCF) the cell pellets were lysed with 500 µL of lysis buffer #4 (30 min RT vortexing). Lysates were diluted 20-fold in 1X lysis buffer and 16 µL of diluted lysate were transferred (triplicates) to a 384 small volume white microtiter plate for Ki-67 HTRF assay. The Ki-67 expression is clearly much higher in non-confluent A549 cells compared to confluent-cells.
Differential Ki-67 protein expression in confluent and non-confluent A549 tumor cells
A549 cells were cultured in two T175 flasks in Ham's F-12 complete medium (10% FCS), using two seeding concentrations to obtain either confluent cells or non-confluent cells. The adherent cells were washed with PBS, then dissociated (5 mL cell dissociation buffer), scraped with a rubber policeman and transferred to three 1.5 mL microtubes (106 cells/tube) as technical replicates. After centrifugation (5 min, 400 RCF) the cell pellets were lysed with 500 µL of lysis buffer #4 (30 min RT vortexing). Lysates were diluted 20-fold in 1X lysis buffer and 16 µL of diluted lysate were transferred (triplicates) to a 384 small volume white microtiter plate for Ki-67 HTRF assay.The Ki-67 expression is clearly much higher in non-confluent A549 cells compared to confluent-cells.
