Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Summary

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

Abstract

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion ^{1, 2}. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues ³ provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.

Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration ². Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.

Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function ⁴, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO₂ supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

Protocol

1. Preparation of Plate Coated with Collagen

1. Dilute collagen to a final concentration of 10 μg/ml in Opti-Mem media.
2. Coat 6 well plates with collagen, by adding 1.5 ml from step 1 to each well. Incubate overnight at 4 °C.
3. On the following day, wash the plate 2 times with 1x Phosphate Buffered Saline (PBS) and store them in PBS.

2. Cell Preparation and Seeding on a 6-well Plate

Note: Use warm media and PBS to ensure optimal conditions for cell movement

1. Grow MDA-MB-231 (an invasive breast cancer cell line) sparsely in Dulbecco's Modified Eagle Medi....

Discussion

The real time random migration assay enables accurate, sensitive, analysis of cell migration parameters such as speed and displacement. This method is not restricted to end point measurement value, hence it is more quantitative. Since, cells are monitored in normal DMEM medium with serum; it reduces the deleterious effect of longer incubation in serum free media. It has been shown that the migration of cells depends on the formatting and breaking cell contacts with the substratum⁸, thus, this method could be u.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	Thermo Fisher Scientific, Inc.	SH30243.01
FBS	Gemini Bio Products	100-106
Opti-Mem	Invitrogen	31985-070
Collagen	BD Biosciences	354231
Microscope with Live Cell chamber & Automated XY stage controller	Olympus Corporation	Olympus 1X81
Environmental control chamber	Neue	Neue Live Cell Chamber	Ours has a custom built rectangular glass plate top for appropriate optics through the top of the chamber. The rectangular shape accommodates multi-well plates used in many of our experiments.
Automated XY stage	Prior Scientific	Prior Proscan	This allows precise return to multiply selected XY positions during time lapse microscopy.
Camera		Hamamatsu EM camera C9100
Software	Typically purchased through microscope vendor such as Olympus	Slide Book 5