Single-cell Microinjection for Cell Communication Analysis

Summary

We describe here how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells, and provide some useful tips. We expect that this paper will help everyone to evaluate the degree of cellular coupling due to functional gap junctions. Everything described here could be, in principle, adapted to other fluorescent dyes with molecular weight below 1,000 Daltons.

Abstract

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a hemichannel by each neighboring cell to form the gap junction. In mammalian cells, the hemichannel is formed by six connexins, monomers with four transmembrane domains and a C and N terminal within the cytoplasm. Gap junctions permit the exchange of ions, second messengers, and small metabolites. In addition, they have important roles in many forms of cellular communication within physiological processes such as synaptic transmission, heart contraction, cell growth and differentiation. We detail how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells. It is expected that in functional gap junctions, the dye will diffuse from the loaded cell to the connected cells. It is a very useful technique to study gap junctions since you can evaluate the diffusion of the fluorescence in real time. We discuss how to prepare the cells and the micropipette, how to use a micromanipulator and inject a low molecular weight fluorescent dye in an epithelial cell line.

Introduction

Gap junctions are intercellular channels that allow the intercommunication among neighboring cells¹. This communication connects two or more neighboring cells, where each one contributes with a connexon or hemichannel to form the intercellular channel. In mammalian cells, the connexon is formed by six connexins, monomers with four transmembrane domains and a C and N terminal within the cytoplasm². Gap junctions not only permit the flow of ions, second messengers and small metabolites, but also contribute to many forms of cellular communication in many physiological processes, such as synaptic transmission, heart contract....

Protocol

1. Preparation of Cells

1. Maintain a culture of a thymic epithelial cell line (IT76M1) or cell to be tested in an incubator (37°C/5% CO₂).
2. Wash the cells with PBS 1x (repeat this item 3x).
3. Add Trypsin to the cells for 5 min.
4. Add medium (twice of the volume of trypsin added in item 1.3) with 10% FBS (fetal bovine serum) to the cells with trypsin and centrifuge (800 x g for 5 min).
5. Count the cells in a hemocytometer.
6. Adjust the density of cells according to the cell type as the cells have to be in close contact with each other to allow coupling. Note: In our case, we used 3 x 10⁵ cell....

Results

Thymic epithelial cell line IT-76MI were used to evaluate dye coupling by gap junctions as these cells were described to express functional gap junctions formed by connexin 43²¹. Figure 1 shows the injection of Lucifer Yellow when applied in the one cell below the tip of the pipette. After few minutes, connected cells become fluorescent (asterisks) indicating the diffusion of the fluorescent dye through the gap junctions. The number of cells and time to became fluo.......

Discussion

In order to verify the presence of functional intercellular gap junction, the use of tracers, which are membrane impermeable, although permeable by intercellular channels are required¹⁶. Fluorescein, the first fluorescent dye to observe cell-to-cell coupling²², is permeable between non junctional membranes³ and has therefore been substituted by Lucifer Yellow dye¹⁵. Currently, to find the best choice among the many different t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Lucifer yellow	Sigma	L0259
Lithium Chloride	Sigma	L4408
PBS tablets	Sigma	P4417
RPMI	Sigma	R4130
Bovine fetal serum	Cultilab
Trypsin	Sigma	T4799
vibration-insulated table	Newport	VH3036W-OPT	A vibration-insulated table is needed to protect the experiments from vibration and avoid cell damage
Micromanipulator	Narishige	MMO-203	This equipment allows precision adjustments of the micropipette, which is needed for cell micro injection.
Current Generator	Digitimer	DS2	To produce the dye flow through the micropipette, a current below one nano ampere was given using a current generator with an electrode inside the micropipette or an amplifier which has a capacitance compensation circuit (old electrometer) or current injection functions of new patch clamp amplifiers, and the ground wire submersed in the plate dish. Alternatively, the dye can be injected by a pneumatic microinjector, following the factory recommendations.