Protocol for Analysis and Consolidation of TrackMate Outputs for Measuring Two-Dimensional Cell Motility using Nuclear Tracking

Summary

This protocol for high-throughput measurement of cell motility in HaCaT keratinocytes describes methods for collecting and processing images of cell nuclei and performing particle tracking using the ImageJ plugin TrackMate.

Abstract

Collective cellular migration plays a key role in many fundamental biological processes including development, wound healing, and cancer metastasis. To understand the regulation of cell motility, we must be able to measure it easily and consistently under different conditions. Here we describe a method for measuring and quantifying single-cell and bulk motility of HaCaT keratinocytes using a nuclear stain. This method includes a MATLAB script for analyzing TrackMate output files to calculate displacements, motility rates, and trajectory angles in single cells and in bulk for an imaging site. This motility analysis script allows for quick, straightforward, and scalable analysis of cell motility rates from TrackMate data and could be broadly used to identify and study the regulation of motility in epithelial cells. We also provide a MATLAB script for reorganizing microscopy videos collected on a microscope and converting them to TIF stacks, which can be analyzed using the ImageJ TrackMate plugin in bulk. Using this methodology to explore the roles of adherens junctions and actin cytoskeletal dynamics in regulating cell motility in HaCaT keratinocytes, we demonstrate evidence that Arp2/3 activity is required for the elevated motility seen after α-catenin depletion in HaCaT keratinocytes.

Introduction

Precise, responsive regulation of cellular motility in epithelial cells is crucial for wound healing and for replenishing the epithelial layer. Failure to sustain motility can lead to problems with embryonic development and wound healing¹ and overactive motility signaling is a key contributor to cancer metastasis².

Understanding cellular control of motility in HaCaT keratinocytes offers important insights into these processes. The procedures outlined here provide consistent measurements and calculations of the average magnitude of cellular motility on a single-cell or population level. We have....

Protocol

1. Cell culture and imaging plate preparation

1. Culture HaCaT cells in cell culture medium (Dulbecco's Modified Eagle's Medium supplemented with 2 mM L-glutamine, 100 U/mL penicillin/ streptomycin, and 10% (v/v) fetal bovine serum) at 37 °C and 5% carbon dioxide in a humidified incubator.
2. Seed desired number of cells per well (we use 5000 - 20,000 cells/well) in a 96-well imaging plate, such as a 96-well black polystyrene microplate. To three wells, add only medium (no cells).
3. Rest the plate on a completely flat surface for 20 - 30 min so that cells can attach to the bottom of the plate with even spatial....

Results

To ensure that our analysis script was reliable and consistent, we measured the motility of HaCaT keratinocytes in three independent experiments. We found that while the standard deviation of cell motilities was variable between experiments (possibly due to the sensitivity of HaCaT cells to confluence and mechanical stimuli), the average motility was consistently replicated across multiple experiments without statistically significant differences between replicates according to a two-tailed student's t-test (

Discussion

The methodology and analysis tools described above provide a straightforward and scalable means for measuring and quantifying the motility of HaCaT keratinocytes that uses MATLAB and requires minimal programming experience. This protocol calls for the nuclei of cells to be stained and imaged over the course of at least 5 h. While data collection can be performed on any suitable microscope, this protocol provides a script for processing images collected on an ImageXpress Micro XL microscope into a TIFF stack for each imag.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
96-well flat clear bottom black polystyrine TC-treated microplates, individually wrapped, with lid, sterile	Corning	3603
Dulbecco's modified eagle medium (high D-glucose)	Life Technologies Corporation/Thermo Fisher Scientific	12800-082
Fetal bovine serum	Sigma-Aldrich Inc	F0926
Fluorobrite Dulbecco's modified eagle medium (high D-glucose, 3.7 g/L sodium bicarbonate, no L-glutamine, no phenol red)	Gibco/Thermo Fisher Scientific	A18967-01
GlutaminePlus	R&D Systems Inc.	R90210
Hoechst 33342, trihydrochloride, trihydrate	Invitrogen/Thermo Fisher Scientific	H21492
Penicillin streptomycin	Life Technologies Corporation/Thermo Fisher Scientific	15140-122
Phosphate buffered saline	Gibco/Thermo Fisher Scientific	14190-144