Journal: Islets

Article Title: Stabilization protects islet integrity during respirometry in the Oroboros Oxygraph-2K analyzer

doi: 10.1080/19382014.2022.2054251

Figure Lengend Snippet: A 3D printed mesh device to stabilize islets within the Oxygraph-2 K chamber. (a) Structural components of the islet containment device are shown. The top cap and bottom cup of the device were printed on a fused deposition 3D printer (Creality3D) using transparent PLA filament. 40 µm nylon mesh was secured to both the cap and the bottom chamber using plastic rings. (b) Wild-type mouse islets were gently pipetted onto the bottom chamber and evaluated on a dissecting microscope (left). Three O-ring rubber sections were fit to the grooves of the cap, and the cap was pressure fitted onto the bottom cup containing the islets (right). (c) The entire device containing islets was loaded into an Oxygraph-2 K chamber (Oroboros) and gently submerged in Mir05 buffer to rest just above the stir bar.

Article Snippet: This investigation used groups of 400–500 islets per condition, which is consistent with sample sizes from prior studies ranging from 150 to 1,000 islets per group., , These large numbers are a stark contrast to Seahorse islet studies where groups of 30–70 islets are often used for 24-well islet capture plates., Oroboros Instruments does offer an alternative Oxygraph chamber (O2k-chamber sV, available in the O2k-sv Module) which reduces the chamber volume from 2.0 ml to 0.5 mL, thereby reducing the sample size needed for respirometry.

Techniques: Microscopy

Journal: Islets

Article Title: Stabilization protects islet integrity during respirometry in the Oroboros Oxygraph-2K analyzer

doi: 10.1080/19382014.2022.2054251

Figure Lengend Snippet: Islet stabilization does not inhibit cellular response to metabolic modulators. (a) Depiction of a standard Oxygraph respirometry trace. Anticipated oxygen fluctuation (curved line) in response to common drug injections (arrows) are shown. Areas of curve stabilization (dashed lines) are used to calculate respiratory parameters from respirometry data. (b) Representative respirometry trace from wild-type mouse islets free in the Oxygraph-2 K chamber containing Mir05 buffer. (c) Representative respirometry trace from islets in a mesh device submerged in Mir05 buffer within the Oxygraph-2 K chamber. Oxygen concentration (blue) and oxygen flux (red) are shown for each experimental trace. Metabolic drugs were sequentially added to each chamber (arrows) to evaluate parameters of mitochondrial respiration; O, Oligomycin; F, FCCP; R, rotenone; A, antimycin A. (d) Representative areas of each oxygen flux trace were selected to calculate basal respiratory rate (Routine), “leak” state, maximal respiratory capacity (OXPHOS), and non-mitochondrial respiration (ROX). Averaged oxygen flux values were normalized to ROX to calculate oxygen flux ratios.

Article Snippet: This investigation used groups of 400–500 islets per condition, which is consistent with sample sizes from prior studies ranging from 150 to 1,000 islets per group., , These large numbers are a stark contrast to Seahorse islet studies where groups of 30–70 islets are often used for 24-well islet capture plates., Oroboros Instruments does offer an alternative Oxygraph chamber (O2k-chamber sV, available in the O2k-sv Module) which reduces the chamber volume from 2.0 ml to 0.5 mL, thereby reducing the sample size needed for respirometry.

Techniques: Concentration Assay

Journal: Islets

Article Title: Stabilization protects islet integrity during respirometry in the Oroboros Oxygraph-2K analyzer

doi: 10.1080/19382014.2022.2054251

Figure Lengend Snippet: Islet stabilization reduces fragmentation. (a) Representative images of wild-type mouse islets imaged before and after free and stabilized respirometry in an Oroboros Oxygraph-2 K system. 10x (middle) and 40x (right) images were collected on a DMI8 Widefield Epifluorescence Microscope (Leica). Particle analysis (left) of free and stabilized islet populations was performed using Image J software. 10X images chosen for analysis represent approximately 11% (before) and 17% (after) of total islet populations. (B) Quantification of particle size in free and stabilized islets both before and after respirometry analysis. Data are mean ± SEM for 3 individual experiments. * P < .05; unpaired t test.

Article Snippet: This investigation used groups of 400–500 islets per condition, which is consistent with sample sizes from prior studies ranging from 150 to 1,000 islets per group., , These large numbers are a stark contrast to Seahorse islet studies where groups of 30–70 islets are often used for 24-well islet capture plates., Oroboros Instruments does offer an alternative Oxygraph chamber (O2k-chamber sV, available in the O2k-sv Module) which reduces the chamber volume from 2.0 ml to 0.5 mL, thereby reducing the sample size needed for respirometry.

Techniques: Microscopy, Particle Size Analysis, Software