Measuring islet oxygen consumption in a high throughput format.

<p>A) Islet plate development. The XF24 measures oxygen consumption from monolayers of adherent cells in a 24 well cell culture plate format. Oxygen consumption is measured with probe heads coated with oxygen sensitive fluorophores that are optically read (marked white and red). During the “measure” mode a volume of only a few micro-liters is formed. In this minute volume oxygen tension rapidly drops which enables OCR calculations. In “mix” mode, the probe head moves up and down, reoxygenating the cells and exposing them to injected compounds. The wells with flat bottoms, originally designed for cell monolayers, proved inadequate for islets as they gathered in the well periphery, too far from the probe head (left panel). To address this problem, an islet plate with a central depression covered by a screen was developed. This trapped the islets while still maintaining sufficient media access (right panel). The screen consists of a polycarbonate ring attached to a nylon net with 50 µm pore size. B) Image of islet plate well. After experiments each well of the 24-well plate was imaged and the images were used for calculating OCR per islet and to measure islet diameter. The islet images were then used to obtain an exact islet count per well and to measure islet diameter. C) Flat bottom (V7) plates are not suitable for islets. 70 islets were seeded per well. Traces recorded from separate wells in a plate are shown. Note the large data variation and that subsequent to 20 mM glucose injection the OCR only increase in one well out of four. D) Islet plate reduces data variability. As islet seeding could vary and thus the absolute OCR, data from each well was often normalized to its initial steady state values before any compound was injected. Traces from islets in 3 mM and 20 mM glucose are shown with absolute (left) and normalized rates (right); note the stability over 2.5 hours. Subsequent to 20 mM glucose injection (after 1^st data point) the OCR increased in all the wells. Islets that stay in 3 mM glucose do not display any major change in OCR. E) Initial measurements are unstable. OCR in the islet plate showed an initial drift during the first 1–2 measurements (dashed square), therefore these data points where always omitted. F) Experimental set up – bioenergetic principles. Respiration recorded may be deciphered by using drugs acting on the mitochondrial inner membrane/complexes. Oligomycin blocks complex V and remaining respiration represents the proton leak. Rotenone/Myxothiazol blocks complexes I/III and remaining respiration is non-mitochondrial. FCCP stimulation shows maximal respiratory capacity. Nutrient stimulation (glucose) may be used in addition to these drugs to study the effect of nutrient metabolism.