December 7 – 11, 2019
Walter E. Washington Convention Center Washington, D.C.
Change can happen in an instant. Find out how the IncuCyte® Live Cell Analysis System, reagents and consumables make it easier than ever to visualize cell behavior and quantify cell function in real time. Derive deeper and more physiologically relevant information about your cells, plus real-time kinetic data, without ever removing your cells from the incubator.
Learn how our other solutions enable you to spend less time harvesting cells and collecting data and more time analyzing your data, gaining insights, and making discoveries. When your discovery develops into a therapy, our instruments will deliver large-scale cultures with transferrable process optimization.
While at the event, stop by our poster presentation. Details below.
Title: Dynamic Live-cell Visualization And Quantification Of Cell Cycle And Pharmacological Response Using A Genetically-Encoded, Ubiquitination-Based Sensor
Date | Time: Monday, November 9, 2019 1:30pm
Location: Exhibit Hall AB
Board Number: B258
Authors: L.A. Skerlos, N.J. Bevan, G.F. Lovell, S. Perez, R.J. Lister, G.S. Filonov, T. Dale, D.M. Appledorn, C.L. Schramm
Essen BioScience, Ann Arbor, MI & Welwyn Garden City, UK
Monitoring the progression or disruption of the cell cycle is a relevant tool for the advancement of cancer and regenerative medicine research. The ability to view and analyze fluctuations in the cell cycle enables researchers to answer key questions around proliferation, toxicity, and mechanism of action of therapeutic compounds. Standard technologies to monitor cell cycle are often limited to end-point analysis. The IncuCyte® Live-Cell Analysis System is uniquely suited to visualize, monitor and analyze cell cycle over time. Here we demonstrate the utility of the IncuCyte® Cell Cycle Red/Green Lentivirus Reagent, encoding a FUCCI (Fluorescence Ubiquitination Cell Cycle Indicator) multi-gene expression system to enable live-cell analysis. The sensor was transduced into a panel of cell lines, resulting in stable, non-perturbing expression of green or red fluorescent proteins during S/G2/M or G1, respectively. The IncuCyte® Cell-by-Cell Analysis Software Module was used to quantify subpopulations of cells within each stage of the cell cycle across multiple cell divisions. To study cell cycle synchronization, HeLa cells were treated with thymidine (2.5 mM, 24 h), a DNA synthesis inhibitor, to arrest cell growth in S/G2/M, resulting in 75% of cells expressing green fluorescence. Following release of the thymidine block, image-based measurements were acquired to track the synchronized progression of the cell population as it passed through three 17-hour cycles, as demonstrated by changes in fluorescence: S/G2/M (green), M/G1 (colorless), G1 (red), and G1/S (yellow). To demonstrate pharmacological disruption of cell cycle, a fibrosarcoma cell line (HT1080) was treated with cisplatin or 5-fluorouracil (5FU), resulting in concentration-dependent arrest of the cell cycle in S/G2/M or G1, respectively. Mechanism of action was demonstrated by treating a panel of breast cancer cell lines with diverse hormone receptor patterns (T47D, ER+ & HR+; AU565, ER- & HER2+; MDA-MB-231, TN) with highly targeted compounds. Tamoxifen binds the estrogen receptor and induced arrest only in T47D, while CB-839 showed activity only in triple negative MDA-MB-231 cells. In an additional study to couple observations of drug-induced cell cycle arrest with immune cell morphology and function, THP-1 monocytes were exposed to Phorbol 12-myristate 13-acetate (PMA; 100 nM). PMA-induced differentiation resulted in a decrease in cell proliferation and increase in cell area, along with an increase in percentage of cells expressing red fluorescence, indicative of cell cycle arrest in G1. Overall, these data highlight the utility of the IncuCyte Cell Cycle Red/Green Lentivirus Reagent to provide valuable kinetic measurement of cell cycle phase distribution by live-cell analysis.