Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

Summary

Live-cell imaging provides a wealth of information on single cells or whole populations that is unattainable by fixed cell imaging alone. Here, live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel are described.

Abstract

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over the past decade, new and innovative technologies have greatly enhanced the practicality of live-cell imaging. Cells can now be kept in focus and continuously imaged over several days while maintained under 37 °C and 5% CO₂ cell culture conditions. Moreover, multiple fields of view representing different experimental conditions can be acquired simultaneously, thus providing high-throughput experimental data. Live-cell imaging provides a significant advantage over fixed-cell imaging by allowing for the direct visualization and temporal quantitation of dynamic cellular events. Live-cell imaging can also identify variation in the behavior of single cells that would otherwise have been missed using population-based assays. Here, we describe live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel. We demonstrate methods to visualize whether mitotically arrested cells die directly from mitosis or slip back into interphase. We also describe how the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system can be used to assess the fraction of interphase cells born from mitotic slippage that are capable of re-entering the cell cycle. Finally, we describe a live-cell imaging method to identify nuclear envelope rupture events.

Introduction

Anti-mitotic drugs have long been used in the chemotherapeutic regimens of various types of solid tumors and often show great efficacy^1,2,3. Mechanistically, these drugs disrupt normal mitotic progression and promote mitotic arrest in rapidly proliferating cancer cells. However, cell fate in response to mitotic arrest is highly variable: while a fraction of cells undergoes cell death directly from mitosis, others exit out of mitosis and return to interphase as tetraploid cells (a process termed mitotic slippage)^4,5

Protocol

This protocol focuses on using the non-transformed and chromosomally stable retinal pigmented epithelial (RPE-1) cell line for live-cell imaging experiments. However, this protocol can be adapted to any adherent cell line so long as cell culture conditions are adjusted as necessary. All procedures must adhere to institutional biosafety and ethical guidelines and regulations.

1. Preparing Cells for Live-cell Imaging

1. Use freshly thawed and early passage RPE-1 cells expressing either human histone H2B fused to a fluorescent protein (e.g. H2B-GFP), or the FUCCI system (a detailed protocol on how to generate FUCCI-ex....

Results

Using the protocol described above, RPE-1 cells expressing H2B-GFP were treated with an anti-mitotic or vehicle control (DMSO) and analyzed by live-cell imaging. Analysis revealed that 100% of control mitotic RPE-1 cells initiated anaphase an average of 22 min after entering mitosis (Figure 1A, 1D). By contrast, RPE-1 cells treated with paclitaxel exhibited a profound mitotic arrest lasting several hours (Figure 1D

Discussion

The most critical aspect of long-term live-cell imaging is ensuring the health of the cells being imaged. It is essential that cells be exposed to minimal extraneous environmental stressors, such as substandard conditions regarding temperature, humidity, and/or CO₂ levels. It is also important to limit photodamage from fluorescent excitation illumination, as imaging stress is known to affect cell behavior⁵⁰. Limiting photodamage can be achieved in multiple ways as detailed elsewhere

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
phenol red-free, DMEM:F12 media	Hyclone	SH3027202
10% fetal bovine serum (FBS)	Gibco	10438026
100 IU/ml penicillin, and 100 mg/ml streptomycin.	Gibco	15070063
PBS	Gibco	10010049
0.25% Trypsin/EDTA	Gemini	50-753-3104
12-well glass bottomed imaging dish	Cellvis	P12-1.5H-N
Paclitaxel	TSZ Chem	RS036
Dimethyl Sulfoxide (DMSO)	Sigma	NC0215085
Environmental Chamber	In Vivo Scientific
5% CO2	Airgas	Z03NI7432000201
15 mL Conical Tubes	Fisherbrand	05-539-12
10 cm polystyrene tissue culture plates	Falcon	08772E
10 mL Disposable Serological Pipette	Fisherbrand	4488
5804R 15 amp Centrifuge	Eppendorf	97007-370
1000 microliters Pipette	Gilson	Pipetman Classic
20 microliters Pipette	Gilson	Pipetman Classic
p1000 Pipette Tips	Sharp	p1126
p20 Pipette Tips	Sharp	P1121
RPE-1 Cells	ATCC	CRL-4000
Incubator Galaxy 170S	Eppendorf	CO17011005
Pipette Boy	Integra	156401
Hemacytometer	Fisher Scientific	0267110
Nikon Ti-E-PFS Inverted Microscope for Live Cell Imaging	Micro Video Instruments, Inc.	MEA53100
Prior X-Y Stage System and White Light LED Illuminator	Micro Video Instruments, Inc.	500-H117P1N4
Prior Proscan 3 Controller XYZ with Encoders	Micro Video Instruments, Inc.	500-H31XYZE
Andor Digital Camera	Micro Video Instruments, Inc.	D10NLC
Nikon NIS Elements AR Software	Micro Video Instruments, Inc.	MQS31000
SOLA LED Illuminator for Fluorescence	Micro Video Instruments, Inc.	SOLA-E
InVivo Environmental Chamber with CO2 Flow Control	Micro Video Instruments, Inc.	Ti-IVS-100
Ti-ND6-PFS Perfect Focus Motorized Nosepiece	Micro Video Instruments, Inc.	MEP59391
Ti-C System Condenser Turret	Micro Video Instruments, Inc.	MEL51000
Ti-C LWD LWD Lens Unit for System Condenser Turret	Micro Video Instruments, Inc.	MEL56200
Te-C LWD Ph1 Module	Micro Video Instruments, Inc.	MEH41100
CFI Plan Fluor DLL 10X Objectivena 0.3 wd 16mm	Micro Video Instruments, Inc.	MRH10101
CFI Super Plan Fluor ELWD 20xc ADM Objective	Micro Video Instruments, Inc.	MRH48230