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Cell Biology JoVE (Open Access) Citable · DOI

Measuring the Induced Membrane Voltage with Di-8-ANEPPS

DOI: 10.3791/1659-v
What you'll learn
  • ✓ Load cells with di-8-ANEPPS potentiometric dye for membrane voltage measurement
  • ✓ Acquire fluorescence images of induced membrane voltage under electric field stimulation
  • ✓ Process and analyze image data to quantify induced membrane voltage (ΔΦ)
Protocol

External electric field induces a voltage on the membrane of a cell, termed the induced membrane voltage (ΔΦ). By using the potentiometric dye di-8-ANEPPS, it is possible to measure the ΔΦ noninvasively. This video shows the protocol for measuring ΔΦ using di-8-ANEPPS.

Difficulty
intermediate
Total time
~1–2 hours per experiment (including dye loading, imaging, and analysis)

Steps

1
Understand induced membrane voltage measurement principles

Learn how external electric fields induce voltage on cell membranes and how di-8-ANEPPS potentiometric dye enables noninvasive detection of this induced membrane voltage (ΔΦ).

▶ 00:06
2
Prepare materials and experimental setup

Set up microscopy equipment, prepare buffers, and arrange dye solutions and cell culture materials for the protocol.

▶ 01:49
3
Load cells with di-8-ANEPPS dye

Incubate cells with di-8-ANEPPS solution to allow cellular uptake of the potentiometric dye, then wash and prepare for imaging.

▶ 02:28
4
Acquire fluorescence images during electric field stimulation

Apply external electric field to cells and capture real-time fluorescence images of di-8-ANEPPS to detect changes in membrane voltage.

▶ 03:21
5
Process images and quantify induced membrane voltage

Analyze fluorescence image data using image processing software to calculate and quantify the induced membrane voltage (ΔΦ) values.

▶ 04:35
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