Evidence map›Paper›PMID 23685703›Full record

ArticleJournal of visualized experiments : JoVE2013

Direct imaging of ER calcium with targeted-esterase induced dye loading (TED).

Samira Samtleben, Juliane Jaepel, Caroline Fecher, Thomas Andreska, Markus Rehberg, Robert Blum

Open access · bronzeAbstract readVideo-Audio Media
PubMed Full text at PMC Publisher
In one paragraph

Article in Journal of visualized experiments : JoVE, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 41 papers.

0numbers the graph read from it
0cells of the map it votes in
41citing papers in PubMed
1.7field-weighted citation impact, top 17% of its field
1 · What the graph read from it

What it found

Each row is one number read from the abstract, on the scale the paper reported it, with its interval. Left of the dashed line favours the treatment, right favours the comparator. Under each row is the sentence it came from. New to these charts? A ten-minute tutorial.

The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.

2 · The registry

The trial behind it

Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.

Neither the registry nor the abstract names a trial number. If this is a trial report, that itself is worth knowing.

3 · Its place in the literature

Who cites it

41 citing papers in PubMed, 60 citations in OpenAlex.

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  13. Shaped by leaky ER: Homeostatic CaFrontiers in physiology · 2022
    Review
  14. Review
  15. Resource for FRET-Based Biosensor Optimization.Frontiers in cell and developmental biology · 2022
    Review
  16. Lipids modulate the BH3-independent membrane targeting and activation of BAX and Bcl-xL.Proceedings of the National Academy of Sciences of the United States of America · 2021
    Article
  17. Article
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4 · The record

Corrections and comments

PubMed lists nothing against this paper. Absence here is not a guarantee, only a check that was made.

5 · Who and what money

Authors and funding

6 authors at 3 institutions in 3 countries.

Samira SamtlebenInstitute for Clinical Neurobiology, University of Wuerzburg.
Juliane Jaepel
Caroline Fecher
Thomas Andreska
Markus Rehberg
Robert Blum
Institute of Neurobiology · BGCentre of Experimental Medicine of the Slovak Academy of Sciences · SKMax Planck Institute of Neurobiology · DE

Funding

No grant is acknowledged in the PubMed record.

6 · The paper itself

Abstract

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca(2+) indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca(2+) indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca(2+) indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca(2+) indicator and a hydrophilic fluorescent dye/Ca(2+) complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F0.

Indexed as

AnimalsCalciumCarboxylesteraseCerebral CortexEndoplasmic ReticulumFluorescent DyesGenetic VectorsHEK293 CellsHeLa CellsHippocampusHumansLentivirusLuminescent ProteinsMiceNeurogliaNeuronsCalciumCarboxylesteraseFluorescent DyesLuminescent ProteinsRecombinant ProteinsRed Fluorescent Protein

Identifiers

PMID23685703
PMCPMC3679584
OpenAlexW2024353892

What OpenQuestion holds

Textmetadata
Read underepoch 390

Registered trials

None linked

Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.