A two-photon FRAP protocol to measure the stereociliary membrane diffusivity in rat cochlear hair cells

Shefin S George; Charles R Steele; Anthony J Ricci

doi:10.1016/j.xpro.2021.100637

A two-photon FRAP protocol to measure the stereociliary membrane diffusivity in rat cochlear hair cells

STAR Protoc. 2021 Jun 30;2(3):100637. doi: 10.1016/j.xpro.2021.100637. eCollection 2021 Sep 17.

Authors

Shefin S George¹, Charles R Steele², Anthony J Ricci^{1

3}

Affiliations

¹ Department of Otolaryngology-Head and Neck Surgery, School of Medicine, 240 Pasteur Drive, Stanford University, Stanford, CA 94305, USA.
² Department of Mechanical Engineering, Building 520, 440 Escondido Mall, Stanford University, Stanford, CA 94305, USA.
³ Department of Molecular and Cellular Physiology, School of Medicine, 300 Pasteur Drive, Stanford University, Stanford, CA 94305, USA.

Abstract

Fluorescence recovery after photobleaching (FRAP) has been widely used to monitor membrane properties by measuring the lateral diffusion of fluorescent particles. This protocol describes how to perform two-photon FRAP on the stereocilia of live cochlear inner hair cells using a lipophilic dye, di-3-ANEPPDHQ, to assess the stereociliary membrane diffusivity. We also detail two-photon FRAP microscope setup and calibration, as well as FRAP parameter setting and data analysis. For complete details on the use and execution of this protocol, please refer to George et al. (2020).

Keywords: Cell Membrane; Microscopy; Molecular/Chemical Probes; Neuroscience.

Publication types

Research Support, N.I.H., Extramural

MeSH terms

Animals
Coloring Agents / metabolism
Diffusion
Fluorescence Recovery After Photobleaching / methods*
Hair Cells, Auditory / metabolism*
Photons
Rats
Stereocilia / metabolism*

Substances

Coloring Agents

Abstract

Publication types

MeSH terms

Substances

Grants and funding