Page 18 - Live-cellanalysis handbook

Live-Cell Analysis Handbook — Third Edition

Kinetic Proliferation Assays

Accurate and reproducible measurements
in a variety of cell culture models

Introduction

Cell proliferation assays are a cornerstone of cancer therapeutic, Live-cell imaging alleviates many of these challenges by allowing
developmental biology, and drug safety research. Analysis of for non-destructive, repeated scanning of the same sample over
the sustained signaling pathways that underlie the progression time using either transmitted or fluorescent imaging modalities.
of tumors, for example, accounts for >12,000 manuscripts in However, there are challenges associated with adopting a live-cell
PubMed, the majority of which use cell proliferation analysis approach for measuring proliferation, such as:
to evaluate tumor cell growth. Despite this, there has not been
a direct, straightforward, scalable method for quantifying cell • Many fluorescent detection reagents perturb cell growth and
proliferation as a continuous event. Rather, the traditional morphology and are therefore unsuitable for kinetic analysis.
approaches are endpoints or at best a series of concatenated
endpoints to measure the time-course. • Imaging more than one sample location requires movement of
the sample, which is particularly problematic for non-adherent
Challenges in monitoring cell proliferation via traditional single cells that can easily move to the edge of the well, or dish, and
end point, non-image based assays that utilize plate readers or cause artifacts.
flow cytometers include:
• Imaging over several cell divisions requires leak-free,
• A single data point may not provide enough information to environmental control systems for temperature, oxygen, and
effectively distinguish impacts of conditions or treatments, (e.g. carbon dioxide that can both maintain the environment and
when comparing early or late acting compounds, or discerning enable access for the operator.
cell-type dependent proliferative effects of drugs).
• Most live-cell imaging platforms require deep operator
• Use of concatenated endpoints utilizes samples from different expertise and are not easily scaled to microplate throughput,
wells that are measured at different points in time. This either due to difficulty with set-up of image acquisition
potentially introduces artifacts, primarily due to variations in or ineffective workflow when viewing and analyzing large
cell seeding densities. numbers of images.

• Many biochemical detection measures (e.g., MTT, LDH, ATP A successful strategy for adoption of a scalable, live-cell
detection) are indirect, destroy the sample, and do not approach for proliferation measurements must address the
represent the true cell number. challenges described above. In this chapter, we illustrate how
proliferation assays using the IncuCyte® Live-Cell Analysis
• Measurements cannot be readily verified visually, and System in conjunction with fit-for-purpose software tools and
morphology changes due to treatment effects cannot be non-perturbing reagents enable kinetic quantification of cell
discerned. proliferation, at microplate scale, for both non-adherent and
adherent cell cultures, both in mono- and co-culture. In addition,
• Proliferation rates of cells grown in co-cultures cannot be cell proliferation measurements can be multiplexed with cell
distinguished. health, morphology, or surface marker measurements.

16