Imaging of changes in sarcoplasmic reticulum [Ca2+] using Oregon Green BAPTA 5N and confocal laser scanning microscopy

White, Carl; McGeown, Graham

doi:10.1054/ceca.2001.0269

Cited by 25 publications

(15 citation statements)

References 34 publications

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“…It has been used to measure Ca 2+ in photoreceptors of invertebrates (99), gastric myocytes (80), cardiac myocytes (100), skeletal muscle fibers (101). …”

Section: ) Low Affinity Ca2+ Indicatorsmentioning

confidence: 99%

Chemical calcium indicators

Paredes

Etzler

Watts

et al. 2008

Methods

502

389

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Our understanding of the underlying mechanisms of Ca 2+ signaling as well as our appreciation for its ubiquitous role in cellular processes and has been rapidly advanced, in large part, due to the development of fluorescent Ca 2+ indicators. In this chapter, we discuss some of the most common chemical Ca 2+ indicators that are widely used for the investigation of intracellular Ca 2+ signaling. Advantages, limitations and relevant procedures will be presented for each dye including their spectral qualities, dissociation constants, chemical forms, loading methods and equipment for optimal imaging. Chemical indicators that are now available allow for intracellular Ca 2+ detection over a very large range (<50 nM to >50 μM). Higher affinity indicators can be used to quantify Ca 2+ levels in the cytosol while lower affinity indicators can be optimized for measuring Ca 2+ in subcellular compartments with higher concentrations. Indicators can be classified into either single wavelength or ratiometric dyes. Both classes require specific lasers, filters, and/or detection methods that are dependent upon their spectral properties and both classes have advantages and limitations. Single wavelength indicators are generally very bright and optimal for Ca 2+ detection when more than one fluorophore is being imaging. Ratiometric indicators can be calibrated very precisely and they minimize the most common problems associated with chemical Ca 2+ indicators including uneven dye loading, leakage, photobleaching and changes in cell volume. Recent technical advances that permit in vivo Ca 2+ measurements will also be discussed.

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“…It has been used to measure Ca 2+ in photoreceptors of invertebrates (99), gastric myocytes (80), cardiac myocytes (100), skeletal muscle fibers (101). …”

Section: ) Low Affinity Ca2+ Indicatorsmentioning

confidence: 99%

Chemical calcium indicators

Paredes

Etzler

Watts

et al. 2008

Methods

502

389

View full text Add to dashboard Cite

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“…This paper should also be consulted for an informed discussion and evaluation of methods. The use of fluorescent probes to measure luminal SR content is discussed in section VIB. Several studies using fluorescent BODIPY linked to ryanodine or thapsigargin and DiOC6 have been used in native smooth muscle: stomach (753), portal vein (221), and uterus (635,636). Recently, the value of BODIPY ryanodine has been questioned as it may not block RyR activity, perhaps due to the presence of the fluorophore (231, 455).…”

Section: Confocal Imaging Of the Srmentioning

confidence: 99%

“…The amount of Ca reported upon blocking SERCA has varied, with substantial depletion of the SR seen in cells from uterus (633), stomach (753), and (cultured) aorta (478, 706) and very little depletion in bladder (218), (toad) gastric (661), and (cultured) uterus (792). These differences are consistent with estimates made in other cell types of leak rates from ϳ10 to 200 mol/min (see Camello et al, Ref.…”

Section: E Sr Ca Leakmentioning

confidence: 99%

Sarcoplasmic Reticulum Function in Smooth Muscle

Wray

Burdyga

2010

Physiological Reviews

154

134

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The sarcoplasmic reticulum (SR) of smooth muscles presents many intriguing facets and questions concerning its roles, especially as these change with development, disease, and modulation of physiological activity. The SR's function was originally perceived to be synthetic and then that of a Ca store for the contractile proteins, acting as a Ca amplification mechanism as it does in striated muscles. Gradually, as investigators have struggled to find a convincing role for Ca-induced Ca release in many smooth muscles, a role in controlling excitability has emerged. This is the Ca spark/spontaneous transient outward current coupling mechanism which reduces excitability and limits contraction. Release of SR Ca occurs in response to inositol 1,4,5-trisphosphate, Ca, and nicotinic acid adenine dinucleotide phosphate, and depletion of SR Ca can initiate Ca entry, the mechanism of which is being investigated but seems to involve Stim and Orai as found in nonexcitable cells. The contribution of the elemental Ca signals from the SR, sparks and puffs, to global Ca signals, i.e., Ca waves and oscillations, is becoming clearer but is far from established. The dynamics of SR Ca release and uptake mechanisms are reviewed along with the control of luminal Ca. We review the growing list of the SR's functions that still includes Ca storage, contraction, and relaxation but has been expanded to encompass Ca homeostasis, generating local and global Ca signals, and contributing to cellular microdomains and signaling in other organelles, including mitochondria, lysosomes, and the nucleus. For an integrated approach, a review of aspects of the SR in health and disease and during development and aging are also included. While the sheer versatility of smooth muscle makes it foolish to have a “one model fits all” approach to this subject, we have tried to synthesize conclusions wherever possible.

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“…Fluorescence imaging and measurement of Ca 2+ concentrations have been reported in numerous papers, using a variety of indicator dyes in different modalities, mostly to assess intracellular concentrations of many different cell types [14, 23, 83], as well as keratinocytes and their organelles (e.g., [4]). Also, advantages and disadvantages comparing Ca 2+ -sensitive dyes and various methodologies for their use have been discussed extensively [35, 40, 41].…”

Section: Introductionmentioning

confidence: 99%

Major translocation of calcium upon epidermal barrier insult: imaging and quantification via FLIM/Fourier vector analysis

et al. 2010

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Calcium controls an array of key events in keratinocytes and epidermis: localized changes in Ca2+ concentrations and their regulation are therefore especially important to assess when observing epidermal barrier homeostasis and repair, neonatal barrier establishment, in differentiation, signaling, cell adhesion, and in various pathological states. Yet, tissue- and cellular Ca2+ concentrations in physiologic and diseased states are only partially known, and difficult to measure. Prior observations on the Ca2+ distribution in skin were based on Ca2+ precipitation followed by electron microscopy, or proton-induced X-ray emission. Neither cellular and/or subcellular localization could be determined through these approaches. In cells in vitro, fluorescent dyes have been used extensively for ratiometric measurements of static and dynamic Ca2+ concentrations, also assessing organelle Ca2+ concentrations. For lack of better methods, these findings together build the basis for the current view of the role of Ca2+ in epidermis, their limitations notwithstanding. Here we report a method using Calcium Green 5N as the calcium sensor and the phasor-plot approach to separate raw lifetime components. Thus, fluorescence lifetime imaging (FLIM) enables us to quantitatively assess and visualize dynamic changes of Ca2+ at light-microscopic resolution in ex vivo biopsies of unfixed epidermis, in close to in vivo conditions. Comparing undisturbed epidermis with epidermis following a barrier insult revealed major shifts, and more importantly, a mobilization of high amounts of Ca2+ shortly following barrier disruption, from intracellular stores. These results partially contradict the conventional view, where barrier insults abrogate a Ca2+ gradient towards the stratum granulosum. Ca2+ FLIM overcomes prior limitations in the observation of epidermal Ca2+ dynamics, and will allow further insights into basic epidermal physiology.

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Imaging of changes in sarcoplasmic reticulum [Ca2+] using Oregon Green BAPTA 5N and confocal laser scanning microscopy

Cited by 25 publications

References 34 publications

Chemical calcium indicators

Chemical calcium indicators

Sarcoplasmic Reticulum Function in Smooth Muscle

Major translocation of calcium upon epidermal barrier insult: imaging and quantification via FLIM/Fourier vector analysis

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