Fluorescence-Based Ratiometric Measurement of CRAC Channel Activity in STIM-Orai-Overexpressing HEK-293 Cells

Methods Mol Biol. 2018:1843:17-39. doi: 10.1007/978-1-4939-8704-7_2.

Abstract

Calcium influx through store-operated Ca2+ entry (SOCE), mediated by STIM-operated Orai channels, is crucial for many cellular functions. To dissect the molecular mechanisms underlying the process of STIM-Orai activation and identify regulators that modify this process, ratiometric imaging of SOCE responses in HEK cells overexpressing STIM and Orai is a routinely used method. Here we describe one commonly used procedure of monitoring SOCE activity with a ratiometric membrane-permeable dye fura-2-AM. Other ratiometric indicators suitable for SOCE measurements are also discussed.

Keywords: Calcium release-activated calcium (CRAC) channel; Fura-2; Fura-4F; Fura-6F; Fura-red; GEM-GECO1; Indo-1; Ratiometric Ca2+ measurements; Store-operated Ca2+ entry (SOCE).

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Calcium / metabolism
  • Calcium Release Activated Calcium Channels / physiology*
  • Calcium Signaling
  • Data Analysis
  • Fluorescent Dyes
  • Gene Expression*
  • HEK293 Cells
  • Humans
  • Image Processing, Computer-Assisted
  • Microscopy, Fluorescence / instrumentation
  • Microscopy, Fluorescence / methods
  • Molecular Imaging* / instrumentation
  • Molecular Imaging* / methods
  • Software
  • Stromal Interaction Molecules / genetics*
  • Stromal Interaction Molecules / metabolism

Substances

  • Calcium Release Activated Calcium Channels
  • Fluorescent Dyes
  • Stromal Interaction Molecules
  • Calcium