Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Summary

Here we present techniques for imaging local IP₃-mediated Ca²⁺ events using fluorescence microscopy in intact mammalian cells loaded with Ca²⁺ indicators together with an algorithm that automates identification and analysis of these events.

Abstract

Cytosolic Ca²⁺ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca²⁺ signaling derives from mechanisms by which Ca²⁺ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca²⁺ microdomains (Ca²⁺ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca²⁺ signals with a 128 x 128 pixel spatial resolution at rates of >500 frames sec^-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca²⁺ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP₃-mediated Ca²⁺ signals in intact mammalian cells loaded with Ca²⁺ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca²⁺ signals. The algorithm localizes sites of Ca²⁺ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Introduction

Calcium ions (Ca²⁺) ubiquitously regulate a diverse range of biological processes, including gene expression, secretion and long-lasting changes in synaptic plasticity¹. One way through which Ca²⁺ can act in such a diverse manner is through the different spatial and temporal patterns of Ca²⁺ signals a cell can generate. For example global elevations in cytosolic [Ca²⁺] trigger contraction in smooth muscle tissue² whereas smaller, localized transient elevations (local Ca²⁺ microdomains) stimulate gene expression essential for learning and memory³.

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Protocol

We present detailed procedures for imaging local Ca²⁺ events in human neuroblastoma SH-SY5Y cells. These procedures can be adapted to image intracellular Ca²⁺ signals in many cell types^8-10.

1. Preparation of Cells

1. Culture the cells to be imaged according to instructions listed on their supplier’s website.
2. A few days prior to imaging, harvest cells grown in a tissue culture flask using 1 ml of 0.25% Trypsin/EDTA (2-3 min). Following detachment, add an equal volume of culture media to inactivate the trypsin.
3. Perform a cell count using a hemocytometer and seed cells into g....

Results

Figure 1A shows a WF image of resting Cal-520 fluorescence in human neuroblastoma SH-SY5Y cells also loaded with ci-IP₃. Exposure of these cells to a 100 msec UV flash to photo-release i-IP₃ elicited transient Ca²⁺ puffs at discrete sites (noted by the white circles in Figure 1A). Fluorescence traces measured at these sites showed a rapid rising phase owing to transient openings of IP₃Rs, followed by a much slower falling phase (Figure .......

Discussion

We describe here protocols for imaging local Ca²⁺ events in cultured mammalian cells using fluorescent Ca²⁺ indicators. Furthermore, we describe an algorithm with an intuitive user interface that automates identification and analysis of acquired data. The procedure described here utilize the fluorescent Ca²⁺ indicator Cal-520, but many other Ca²⁺ sensitive dyes such as Fluo-3, Fluo-4, Fluo-8 and Oregon Green BAPTA-1 perform sufficiently well to image Ca²⁺ microdomain.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Human Neuroblastoma SH-SY5Y cells	ATCC	CRL-2266
Cal-520/AM	AAT Bioquest Inc.	21130
ci-IP3 (D-23-O-Isopropylidene-6-O-(2-nitro-4,5-dimethoxy)benzyl-myo-Inositol 1,4,5-trisphosphate-Hexakis(propionoxymethyl) Ester	SiChem	cag-iso-2-145-10
DMSO/20% pluronic F127	Invitrogen	P-3000MP
EGTA/AM	Invitrogen	E-1219
35 mm glass-bottom imaging dishes	MatTek	P35G-1.5-14-C