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Medicine

Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

Published: May 14th, 2016

DOI:

10.3791/53994

1Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder

Here, we describe a method of long-term time-lapse microscopy to longitudinally track single cells in response to anti-cancer therapeutics.

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing factor in the overall outcome. Immunoblotting, flow cytometry and fixed cell experiments are often used to study how cells respond to anti-cancer drugs. These methods are important, but they have several shortcomings. Variability in drug responses between cancer and normal cells, and between cells of different cancer origin, and transient and rare responses are difficult to understand using population averaging assays and without being able to directly track and analyze them longitudinally. The microscope is particularly well suited to image live cells. Advancements in technology enable us to routinely image cells at a resolution that enables not only cell tracking, but also the observation of a variety of cellular responses. We describe an approach in detail that allows for the continuous time-lapse imaging of cells during the drug response for essentially as long as desired, typically up to 96 hr. Using variations of the approach, cells can be monitored for weeks. With the employment of genetically encoded fluorescent biosensors numerous processes, pathways and responses can be followed. We show examples that include tracking and quantification of cell growth and cell cycle progression, chromosome dynamics, DNA damage, and cell death. We also discuss variations of the technique and its flexibility, and highlight some common pitfalls.

Live-cell microscopy and longitudinal tracking of single cells is not a new technique. From the earliest microscopes, enthusiasts and scientists have observed and studied single cells and organisms, their behaviors, and development1-3. A famous example from the late David Rogers at Vanderbilt University in the 1950s shows a human neutrophil in a blood smear chasing a Staphylococcus aureus bacterium and eventually the process of phagocytosis4. This live-cell movie is an excellent illustration of how multiple processes can be observed and correlated in a single experiment: sensing of a chemical gradient, mechanics and speed of cell motilit....

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 The following protocol uses parameters defined by the experiments in Figures 4 and 6 regarding acquisition settings and experimental conditions. Many of these parameters can be modified to fit other experiments (i.e., exposure times, binning, fluorescent channels, etc.). All procedures must adhere to institutional guidelines and regulations and be approved by the institutional biosafety committee. Microscope manufacturer websites contain excellent information for .......

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Long-term time-lapse microscopy and direct longitudinal tracking allows for the study of many anti-cancer effects during drug response. Following the general outline in Figure 1, multiple examples of cells are shown expressing validated fluorescent reporters that treated with anti-cancer drugs, tracked, and analyzed using different approaches.

Phase contrast microscopy alone is very informative and robustly repo.......

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Advantages of Time-lapse Microscopy and Longitudinal Tracking

The microscope is an ideal instrument for longitudinal studies of drug response as it allows investigators to track individual cells and their fates as well as the entire population. Variability in drug response within a population of cells is a major issue for anti-cancer therapeutic design. Longitudinal tracking of single cells allows investigators to observe this variability and begin to understand the underlying.......

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We thank Joshua Marcus for technical support and Jolien Tyler, Ph.D., Director of the Richard J. McIntosh Light Microscopy Core Facility, for technical advice. This work was supported by funds from the University of Colorado Boulder and the University of Colorado Boulder Graduate School to J.D.O. R.T.B. is partially supported by pre-doctoral training grant from the NIH (T32 GM008759). We thank Karyopharm Therapeutics, Inc. for selinexor and Merck Serono for Kinesin-5 inhibitor. FUCCI plasmids are from Atsushi Miyawaki (RIKEN, Japan) via MTA. mCherry-BP1-2 was from Addgene. HeLa expressing H2b-mCherry and β-tubulin-EGFP are from Daniel Gerlich (IMBA, Austrian Acad....

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Name Company Catalog Number Comments
Taxol (paclitaxel) Sigma T7191 microtubule stabilizing drug
etoposide Selleckchem S1225 topoisomerase II inhibitor
selinexor Karyopharm Therapeutics na XPO1/CRM1 inhibitor, gift
Kinesin-5 inhibitor Merck Serono na gift, also available from American Custom Chemicals Corporation. CAS 858668-07-2
cell growth medium HyClone (Fisher) or Mediatech many companies available
5% CO2/balance air, certified Airgas Z03NI7222004379
35mm dish, 20mm glass bottom Cellvis D35-20-1.5-N many companies available
35mm 4 well dish, 20mm glass bottom Cellvis D35C4-20-1.5-N many companies available
35mm dish, gridded glass bottom MatTek P35G-2-14-CGRD many companies available
multi-well, glass bottom Cellvis P12-1.5H-N many companies available
Olympus IX81 inverted epifluorescence microscope Olympus
Olympus IX2-UCB controller Olympus
PRIOR LumenPro200 Prior Scientific Lumen200PRO
PRIOR Proscan III motorized stage Prio Scientific H117
STEV chamber InVivo Scientific STEV.ECU.HC5 STAGE TOP
Environmental Controller Unit InVivo Scientific STEV.ECU.HC5 STAGE TOP
Hamamatsu ORCA R2 CCD with controller Hamamatsu C10600
Nikon Eclipse Ti Nikon
Nikon laser launch Nikon
SOLA light engine lumencor
iXon Ultra 897 EM-CCD ANDOR 
TOKAI HIT inclubation chamber TOKAI HIT TIZSH

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