Encapsulation and perfusion of mitochondria in agarose beads for functional studies with <sup>31</sup>P‐NMR spectroscopy

Doliba, Nicolai M.; Wehrli, Suzanne; Babsky, Andriy M.; Doliba, Nataliya; Osbakken, Mary D.

doi:10.1002/mrm.1910390502

Cited by 14 publications

(9 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Formation of beads. Cells were placed in agarose beads (bead size 800 -1,000 m; 2000 -2500 beads/cm 3 ) to maintain them in a stable environment and prevent them from escaping from the superfusion system (24). Briefly, cells were suspended in 1 ml Krebs-Ringer bicarbonate buffer containing: 114 mmol/l NaCl, 5 mmol/l KCl, 24 mmol/l NaHCO 3 , 1 mmol/l MgCl 2 ϫ 6H 2 O, 25 mmol/l creatine, 2.2 mmol/l CaCl 2 , 10 mmol/l HEPES (pH 7.4), and 3 mmol/l glucose.…”

Section: Methodsmentioning

confidence: 99%

Differential Effects of Glucose and Glyburide on Energetics and Na+ Levels of βHC9 Cells

et al. 2003

Self Cite

View full text Add to dashboard Cite

In the present study, noninvasive 31 P and 23 Na ؉ -nuclear magnetic resonance (NMR) technology and respirometry were used to compare the effect of high glucose (30 mmol/l) with the effect of the antidiabetic sulfonylurea (SU) compound glyburide (GLY) on energy metabolism, Na ؉ flux, insulin, and cAMP release of continuously superfused ␤-HC9 cells encapsulated in microscopic agarose beads. Both high glucose and GLY increased oxygen consumption in ␤-HC9 cells (15-30%) with a maximal effect at 8 mmol/l for glucose and at 250 nmol/l for GLY. At the same time, insulin release from ␤-cells increased by 15-and 25-fold with high glucose or GLY, respectively. The P-creatine (PCr) level was greatly increased and inorganic phosphate (P i ) was decreased with 30 mmol/l glucose in contrast to the decreased level of PCr and increased P i with GLY. ATP levels remained unchanged during both interventions. Studies on isolated mitochondria of ␤-HC9 cells showed that GLY added to mitochondria oxidizing glutamine or glutamate abolished the stimulation of respiration by ADP (state 3) meanwhile leaving state 3 respiration unchanged during oxidation of other substrates. Exposure of ␤-HC9 cells to 5 mmol/l glucose decreased intracellular Na ؉ levels monitored by 23 Na ؉ -NMR spectroscopy and 30 mmol/l glucose resulted in a further decrease in cytosolic Na ؉ . In contrast, Na ؉ increased when 1 mol/l GLY was added to the perfusate containing 5 mmol/l glucose. These data support the hypothesis that glucose activates the ␤-cell through a "push mechanism" due to substrate pressure enhancing fuel flux, energy production, and extrusion of Na ؉ from the cells in contrast to SU receptor (SUR)-1 inhibitors, which may modify intermediary and energy metabolism secondarily through a "pull mechanism" due to higher energy demand resulting from increased ion fluxes and the exocytotic work load. Diabetes 52:394 -402, 2003

show abstract

Section: Methodsmentioning

confidence: 99%

Differential Effects of Glucose and Glyburide on Energetics and Na+ Levels of βHC9 Cells

et al. 2003

Self Cite

View full text Add to dashboard Cite

show abstract

“…Fabrication of the microsphere and the fiber taper is an inexpensive process and involves commercially available fibers. Agarose gel is well known as a hygroscopic polymer commonly used in biological research [20,21], with an advantage of better long-term stability compared with materials used in [22] and [23]. Three sensor samples based on the silica microspheres dip-coated with Agarose gel of different concentrations (0.5, 1.125 and 2.25 wt./vol.…”

Section: Introductionmentioning

confidence: 99%

Agarose coated spherical micro resonator for humidity measurements

Mallik¹,

Liu²,

Kavungal³

et al. 2016

Opt. Express

View full text Add to dashboard Cite

Abstract:A new type of fiber optic relative humidity (RH) sensor based on an agarose coated silica microsphere resonator is proposed and experimentally demonstrated. Whispering gallery modes (WGMs) in the micro resonator are excited by evanescent coupling using a tapered fiber with ~3.3 µm waist diameter. A change in the relative humidity of the surrounding the resonator air induces changes in the refractive index (RI) and thickness of the Agarose coating layer. These changes in turn lead to a spectral shift of the WGM resonances, which can be related to the RH value after a suitable calibration. Studies of the repeatability, long-term stability, measurement accuracy and temperature dependence of the proposed sensor are carried out. The RH sensitivity of the proposed sensor depends on the concentration of the agarose gel which determines the initial thickness of the deposited coating layer. Studies of the micro-resonators with coating layers fabricated from gels with three different Agarose concentrations of 0.5%, 1.125% and 2.25 wt./vol.% showed that an increase in the initial thickness of the coating material results in an increase in sensitivity but also leads to a decrease of quality factor (Q) of the micro resonator. The highest sensitivity achieved in our experiments was 518 pm/%RH in the RH range from 30% to 70%. The proposed sensor offers the advantages of a very compact form factor, low hysteresis, good repeatability, and low cross sensitivity to temperature.

show abstract

“…11,12 Furthermore, dead cells are rapidly depleted of their ADP and ATP. 6,8,9,13 If dead cells are rapidly depleted of their ADP and ATP content, then the ADP/ATP ratio would be incapable of accounting for any dead cells found within an islet preparation and would be effectively overestimating the viability of the preparation.…”

mentioning

confidence: 99%

The ATP/DNA Ratio Is a Better Indicator of Islet Cell Viability Than the ADP/ATP Ratio

Suszynski¹,

Wildey²,

Falde³

et al. 2008

Transplantation Proceedings

View full text Add to dashboard Cite

Real-time, accurate assessment of islet viability is critical for avoiding transplantation of nontherapeutic preparations. Measurements of the intracellular ADP/ATP ratio have been recently proposed as useful prospective estimates of islet cell viability and potency. However, dead cells may be rapidly depleted of both ATP and ADP, which would render the ratio incapable of accounting for dead cells. Since the DNA of dead cells is expected to remain stable over prolonged periods of time (days), we hypothesized that use of the ATP/DNA ratio would take into account dead cells and may be a better indicator of islet cell viability than the ADP/ATP ratio. We tested this hypothesis using mixtures of healthy and lethally heat-treated (HT) rat insulinoma cells and human islets. Measurements of ATP/DNA and ADP/ATP from the known mixtures of healthy and HT cells and islets were used to evaluate how well these parameters correlated with viability. The results indicated that ATP and ADP were rapidly (within 1 hour) depleted in HT cells. The fraction of HT cells in a mixture correlated linearly with the ATP/DNA ratio, whereas the ADP/ADP ratio was highly scattered, remaining effectively unchanged. Despite similar limitations in both ADP/ADP and ATP/ DNA ratios, in that ATP levels may fluctuate significantly and reversibly with metabolic stress, the results indicated that ATP/DNA was a better measure of islet viability than the ADP/ATP ratio.Islet cell transplantation is emerging as a promising therapy for the treatment of type 1 diabetes. [1][2][3][4][5] Despite recent advances, the transplantation of islets poses a unique challenge with respect to achieving a consistent clinical outcome. Part of this challenge is being able to reliably and rapidly assess clinical islet quality through the quantification of viability and function before transplantation. Current viability assays are limited in their ability to accurately predict transplantation outcomes in vivo. 6 Consequently, to improve the clinical islet transplantation outcomes, it is imperative to develop more accurate viability assays.A proposed method to assess islet viability and potency before transplantation is quantification of the ADP/ATP ratio. 6 -10 This ratio has been specifically applied in discriminating islet preparations that are suitable for clinical transplantation from those that are not. 6 However, this application may be problematic under certain conditions. Intracellular ADP and ATP levels fluctuate rapidly because these high-energy phosphate molecules are rapidly produced and consumed in many intracellular biochemical reactions. It is widely known that viable cells turn over entire ATP stores on the order of minutes, and that dead cells are incapable of replenishing To better account for dead cells and to more accurately assess the viability of an islet preparation, we suggest the use of an ATP/DNA ratio. DNA does not degrade as rapidly as ADP or ATP in a dead cell. Therefore, using direct measurements of DNA, dead cells in an islet preparation...

show abstract

Encapsulation and perfusion of mitochondria in agarose beads for functional studies with ³¹P‐NMR spectroscopy

Cited by 14 publications

References 17 publications

Differential Effects of Glucose and Glyburide on Energetics and Na+ Levels of βHC9 Cells

Differential Effects of Glucose and Glyburide on Energetics and Na+ Levels of βHC9 Cells

Agarose coated spherical micro resonator for humidity measurements

The ATP/DNA Ratio Is a Better Indicator of Islet Cell Viability Than the ADP/ATP Ratio

Contact Info

Product

Resources

About

Encapsulation and perfusion of mitochondria in agarose beads for functional studies with 31P‐NMR spectroscopy

Cited by 14 publications

References 17 publications

Differential Effects of Glucose and Glyburide on Energetics and Na+ Levels of βHC9 Cells

Differential Effects of Glucose and Glyburide on Energetics and Na+ Levels of βHC9 Cells

Agarose coated spherical micro resonator for humidity measurements

The ATP/DNA Ratio Is a Better Indicator of Islet Cell Viability Than the ADP/ATP Ratio

Contact Info

Product

Resources

About

Encapsulation and perfusion of mitochondria in agarose beads for functional studies with ³¹P‐NMR spectroscopy