Measurement of the Intracellular Calcium Concentration with  Fura-2 AM Using a Fluorescence Plate Reader

Martínez, Magdiel; Martínez, Namyr A.; Silva, Walter I.

doi:10.21769/bioprotoc.2411

Cited by 18 publications

(14 citation statements)

References 2 publications

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“…Then, the islets or cells were transferred to KRB buffer with 20 mM glucose and 0.5% BSA. The calcium influx was measured using Tecan Infinite M200 microreader (Tecan Trading, model: Infinite M200) as previously described ( Martínez et al, 2017 ). The data was calculated using the average of first 9 cycles (unstimulated) as baseline.…”

Section: Methodsmentioning

confidence: 99%

Amino acids-Rab1A-mTORC1 signaling controls whole-body glucose homeostasis

et al. 2021

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SUMMARY Rab1A is a small GTPase known for its role in vesicular trafficking. Recent evidence indicates that Rab1A is essential for amino acids (aas) sensing and signaling to regulate mTORC1 in normal and cancer cells. However, Rab1A’s in vivo function in mammals is not known. Here, we report the generation of tamoxifen (TAM)-induced whole body Rab1A knockout (Rab1A −/− ) in adult mice. Rab1A −/− mice are viable but become hyperglycemic and glucose intolerant due to impaired insulin transcription and β-cell proliferation and maintenance. Mechanistically, Rab1A mediates AA-mTORC1 signaling, particularly branched chain amino acids (BCAA), to regulate the stability and localization of the insulin transcription factor Pdx1. Collectively, these results reveal a physiological role of aa-Rab1A-mTORC1 signaling in the control of whole-body glucose homeostasis in mammals. Intriguingly, Rab1A expression is reduced in β-cells of type 2 diabetes (T2D) patients, which is correlated with loss of insulin expression, suggesting that Rab1A downregulation contributes to T2D progression.

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Section: Methodsmentioning

confidence: 99%

Amino acids-Rab1A-mTORC1 signaling controls whole-body glucose homeostasis

et al. 2021

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“…Calcium levels were measured with fura-2 acetoxymethyl ester (AM) (Molecular Probes) following the methodology reported by Refs. 29 , 30 with some modifications. Bacterial cells 1 × 10 7 CFU/mL were incubated with 5 ppm of ARGIRIUM-SUNCs or 30% v/v of H 2 O 2 (control samples) for 2 h at 37 °C.…”

Section: Methodsmentioning

confidence: 99%

The membrane depolarization and increase intracellular calcium level produced by silver nanoclusters are responsible for bacterial death

Molina-Hernández

Aceto

Bucciarelli

et al. 2021

Sci Rep

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This work highlights how our silver ultra nanoclusters (ARGIRIUM-SUNc) hand-made synthesized, are very useful as a bactericide and anti-biofilm agent. The Argirium-SUNc effective antibacterial concentrations are very low (< 1 ppm) as compared to the corresponding values reported in the literature. Different bacterial defense mechanisms are observed dependent on ARGIRIUM-SUNc concentrations. Biochemical investigations (volatilome) have been performed to understand the pathways involved in cell death. By using fluorescence techniques and cell viability measurements we show, for the first time, that membrane depolarization and calcium intracellular level are both primary events in bacteria death. The ARGIRIUM-SUNc determined eradication of different biofilm at a concentration as low as 0.6 ppm. This suggests that the effect of the nanoparticles follows a common mechanism in different bacteria. It is highly probable that the chemical constitution of the crosslinks could be a key target in the disrupting mechanism of our nanoparticles. Since the biofilms and their constituents are essential for bacterial survival in contact with humans, the silver nanoparticles represent a logical target for new antibacterial treatments.

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“…Calcium concentrations were determined using the fura-2-acetoxymethyl ester (fura-2 AM) radiometric dye . Briefly, DU-145 prostate cancer cells seeded in a black 96-well plate (1 × 10 4 cells/well) were treated for 5 h with corchorusoside C ( 1 ), digoxin (0–50 μM, positive control), or DMSO/water (negative control).…”

Section: Methodsmentioning

confidence: 99%

Caspase-Dependent Apoptosis in Prostate Cancer Cells and Zebrafish by Corchorusoside C from Streptocaulon juventas

Anaya-Eugenio¹,

Addo²,

Ezzone³

et al. 2019

J. Nat. Prod.

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Corchorusoside C (1), isolated from Streptocaulon juventas collected in Vietnam, was found to be non-toxic in a zebrafish (Danio rerio) model and to induce cytotoxicity in several cancer cell lines with notable selective activity against prostate DU-145 cancer cells (IC 50 0.08 μM). Moreover, corchorusoside C induced DU-145 cell shrinkage and cell detachment. In CCD-112CoN colon normal cells, 1 showed significantly reduced cytotoxic activity (IC 50 2.3 μM). A preliminary mechanistic study indicated that 1 inhibits activity and protein expression of NF-κB (p50 and p65), IKK (α and β) and ICAM-1 in DU-145 cells. ROS concentrations increased at 5 h posttreatment and MTP decreased in a dose-dependent manner. Moreover, decreased protein expression of Bcl-2 and increased expression of PARP-1 was observed. Furthermore, corchorusoside C increased both the activity and protein levels of caspases 3 and 7. Additionally, 1 induced sub-G1 population increase of DU-145 cells and modulated caspases in zebrafish with non-differential morphological effects. Therefore, corchorusoside C (1) induces apoptosis in DU-145 cells and targets the same pathways both in vitro and in vivo in zebrafish. Thus, the use of zebrafish assays seems worthy of wider application than is currently employed for the evaluation of potential anticancer agents of natural origin.

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Measurement of the Intracellular Calcium Concentration with Fura-2 AM Using a Fluorescence Plate Reader

Cited by 18 publications

References 2 publications

Amino acids-Rab1A-mTORC1 signaling controls whole-body glucose homeostasis

Amino acids-Rab1A-mTORC1 signaling controls whole-body glucose homeostasis

The membrane depolarization and increase intracellular calcium level produced by silver nanoclusters are responsible for bacterial death

Caspase-Dependent Apoptosis in Prostate Cancer Cells and Zebrafish by Corchorusoside C from Streptocaulon juventas

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