Total Cyclin D1 assay principle

The Total Cyclin D1 assay quantifies the expression level of Cyclin D1 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total Cyclin D1 assay uses two labeled antibodies, one coupled to a donor fluorophore and the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In the presence of Cyclin D1 in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein's expression under a no-wash assay format.

Total Cyclin D1 two-plate assay protocol

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a low volume detection plate before the addition of Total Cyclin D1 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Total Cyclin D1 one-plate assay protocol

Detection of Total Cyclin D1 with HTRF reagents can be performed in a single plate used for culturing, treatment, and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.

Induction of Cyclin D1 degradation in breast cancer cells treated with Everolimus

The human breast cancer cell line MCF7 was plated in complete culture medium in a 96-well culture-treated plate under 100,000 cells/well, and incubated overnight at 37 °C-5% CO2. The cells were treated with increasing concentrations of Everolimus for another overnight incubation, and then lysed with 50 µL of supplemented lysis buffer #1 (1X) for 30 minutes at RT under gentle shaking. For the detection step, 16 µL of cell lysate were transferred into a low volume white microplate and 4 µL of the Total Cyclin D1 detection reagents were added. The HTRF signal was recorded after an overnight incubation.

Cell treatment with the mTORC1 inhibitor Everolimus induced a dose-dependent decrease in the cellular content of Cyclin D1, caused by its degradation. These results are in accordance with the literature (Chen et al., Am J Physiol Cell Physiol 317: C244–C252, 2019).

HTRF total Cyclin D1 assay compared to Western Blot

MCF7 cells were cultured in a T175 flask in complete culture medium at 37°C-5% CO2. After a 48h incubation, the cells were treated with 10 µM MG132 for 4h, and then lysed with 3 mL of supplemented lysis buffer #1 (1X) for 30 minutes at RT under gentle shaking.

Serial dilutions of the cell lysate were performed using supplemented lysis buffer, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF Total Cyclin D1 detection reagents. Equal amounts of lysates were used for a side-by-side comparison between HTRF and Western Blot.

Using the HTRF Total Cyclin D1 assay, 400 cells/well were enough to detect a significant signal, while 12,500 cells were needed to obtain a minimal chemiluminescent signal using Western Blot. Therefore in these conditions, the HTRF Total Cyclin D1 assay was 32 times more sensitive than the Western Blot technique.