A Microfluidic Device for Studying Multiple Distinct Strains

Summary

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

Abstract

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

Introduction

Cells are constantly reacting to a dynamically changing environment, by changing their metabolism, transcriptional profile and cellular functions. To study these phenomena, methods that can quantify such changes are needed. One type of readout for such responses is the change in localization of stress related transcription factors in response to nutritional stress.

Microfluidic devices¹ have been used to dynamically manipulate environmental conditions to cells^2-4. They present several advantages for live cell imaging: the environment of the cells can be precisely controlled and dynamically changed; cells can be live-i....

Protocol

1. Creating a Scotch-tape Master⁵

1. Draw or print out desired microchannel layout, to scale, on paper. In our case the design consists of two Y-shaped channels, each 3 mm wide (Figure 2).
2. Cover a glass-slide with layers of scotch tape. The number of layers will determine the height of the channel (about 60 μm per layer). We used 3 scotch tape layers.
3. Place the layout design on a flat surface. Align the slide over the design pattern. Carefully cut the tape on the glass slide with a scalpel according to the layout.
4. Remove the Scotch tape from all regions of the glass slide except those in the layout ....

Results

To demonstrate the separation between different strains we imaged two distinguishable yeast strains in alternate wells. Imaging the full wells shows no cell leakage between wells (Figure 5a,b). Both strains have the transcription factor MSN2 tagged with YFP. To test the simultaneous effect of dynamically changing conditions, we switched the flow rates in the two input channels, creating a step of no-glucose medium, which resulted in localization of Msn2-YFP to the nucleus (Figure 5c,d). .......

Discussion

In this paper we present a simple benchtop method for creating a microfluidic device that allows following several yeast strains simultaneously under dynamic conditions. Following several strains in one channel provides a reliable tool for comparing dynamics of single cell responses in multiple strains. One advantage of our approach is the ability to fabricate the device with simple techniques without the need for a clean room. In fact, we have also carried out the protocol skipping the plasma treatment operations (in st.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
PDMS- SYLGARD 184	Dow Corning USA
Vacuum desiccator	Nalgene	5310-0250
Biopsy punchers	Ted Pella Inc.	Harris Uni-Core 15076 (2 mm), 15074 (1.2 mm)
Syringe pumps	Chemyx	Fusion 200
Corona treater	Electro-technic products	BD-20
Tygon tubing	Tygon	S-54-HL
Concanavalin-A	Sigma	C7275
Scotch tape	3M Scotch	Transparent Tape 1/2"