Authors: Shyam Patel, Sharon Pine & Pranela Rameshwar
Time-lapse video microscopy is a technique that allows one to assess cellular behavior in real-time. Two-dimensional image data is repeatedly collected at distinct time intervals, and a movie is assembled. Applications include assessment of cell migration, division, communication, growth, and death. A unique advantage to time-lapse video microscopy for assessment of these parameters is that data is collected at the single-cell level, allowing one to assess heterogeneity within a mixed population of cells. Therefore, this is particularly advantageous for cancer biology and stem cell biology. In this protocol, we describe the use of time-lapse video microscopy for (1) the real-time monitoring of cell cycle kinetics of heterogeneous populations of cancer cells, and (2) the real-time assessment of symmetric versus asymmetric stem cell division.
This assay will take approximately 96 hours.
Steps 5 and 8d: Phenol red in media will interfere with acquisition of fluorescence images.
Step 8a and 8b: Note carefully for differences in cell cycle times of daughters. The assignment of a particular doubling time designating symmetric vs. asymmetric division should be based on the particular tissue of origin. For example, a difference in daughter cell cycle lengths of 8 hours may be appropriate for breast cancer cells.
Typical mammalian cancer cells divide every 20-24 hours. These cells are rapid cycling, but since they lack self-renewal ability, they will likely cease to divide after five to ten divisions. These individual cells are expected to become senescent with time. This dying process can be seen by real-time visualization and should be noted. On the contrary, stem-like cancer cells are expected to have the longest doubling times and slowest rates of division. These stem-like cells may divide symmetrically or asymmetrically. Under conditions of low confluency and minimal cell-cell contact, it is expected that a high proportion of cells will divide symmetrically, resulting in two identical daughter stem cells, in an effort to repopulate. As confluency increases and cell-cell contact develops, it is expected that there will be an increase in the frequency of asymmetrically dividing cells.
We would like to thank Dr. Sharon Pine from the Cancer Institute of New Jersey for her advice on this assay.
Delineation of breast cancer cell hierarchy identifies the subset responsible for dormancy. Shyam A. Patel, Shakti H. Ramkissoon, Margarette Bryan, Lillian F. Pliner, Gabriela Dontu, Prem S. Patel, Sohrab Amiri, Sharon R. Pine, and Pranela Rameshwar. Scientific Reports 2 () 30/11/2012 doi:10.1038/srep00906
Shyam Patel, Sharon Pine & Pranela Rameshwar, Rameshwar Lab
Correspondence to: Pranela Rameshwar ([email protected])
Source: Protocol Exchange (2013) doi:10.1038/protex.2013.022. Originally published online 11 February 2013.