Preparation of SUIT chemicals describes the preparation of chemicals used in Substrate-Uncoupler-Inhibitor Ttitration (SUIT) protocols.
Communicated by Spitzer Gudrun 2021-03-19
- The chemicals used in mitochondrial studies have to be of highest purity grade possible. Functional activity of mitochondria, whether in their natural cytosolic habitat or in experimental medium, are highly sensitive to fluctuations of the environmental ionic content.
- Most stock solutions are prepared in concentrations that allow for low titration volumina between 1 and 10 µL to avoid unnecessary dilution of the experiment.
- The pH of stock solutions should be adjusted to pH 7. However, if not easily feasible (e.g. Pyruvate has to be prepared freshly every day in a volume of only 200 µL), a deviation from pH 7 is acceptable as long as the titration volume is low and the experimental medium is bufferd.
- The standard process to prepare a stock solution is to weigh the substance, transfer it to a volumetric glass flask and adjust the volume of the solvent to a specific value. Only in some cases (e.g. when a small volume has to be prepared fresh for every experiment like Pyruvate or if the compound is very toxic like Azide or expensive, the solvent is added instead of adjusted. For some compounds (P, Gp, Glu, Azd) the correct volume to be added was experimentally determined 2021-02 (see Volume of the solute).
- Read SDS (safety data sheets) before using toxic chemicals.
- The use of appropriately diluted stock solutions is critical for concentration-dependent substrate-uncoupler-inhibitor kinetic (SUIK) titrations.
- If possible, compounds are solved in water of high purity grade. For example, in Oroboros lab we use Evoqua Water TechnologiesGmbH: deionized ultra-pure water(Ultra ClearTMTP UV UF TM).
- The stock solutions of chemicals dissolved in organic solvents like ethanol and DMSO should be prepared at highest concentrations possible, since the final concentration of solvents in the chamber should be as low as possible.
- Nevertheless, the effects of solvent on mitochondrial function and experimental sensors (ion selective electrodes) have to be evaluated in chemical background tests and control experiments.
- Ethanol used as solvent is meant to be high purity grade, abbreviated as 'ethanol abs.'
- A special care should be taken with the chemicals dissolved in ethanol, as there is a problem of evaporation and subsequent increase of concentration of stock solutions.
- Disadvantage of DMSO: freezing point of +18.55 °C (65.4 °F). When using DMSO as solvent, make sure to bring the stock solution to room temperature before filling the Hamilton syringe, to avoid blocking the needle.
- Most of the chemicals are stable and stored as refrozen aliquots of stock solutions for a long-term usage. However, some chemicals are prone to easy oxidation and have to be prepared freshly for each experiment just prior to adding to the chamber; e.g. pyruvate, α-ketoglutarate, oxaloacetate. For such chemicals, recommendations are given how to prepare the small-volume solutions while attaining a precise stock concentration.
- In Oroboros-lab, all aliquots that are not prepared freshly are stored in the freezer at -20 °C.
- It is advisable to divide stock solutions in small portions before freezing them at -20°C instead of storing the whole amount of stock solution in one vial. Thereby the risk of contamination or water condensation and the number of freezing-thawing-cycles is lower.
- In gerenal, transparent Eppendorf tubes are used to store aliquoted stock solutions. Exceptions:
- light sensitive compounds are stored in dark glass or dark Eppendorf tubes.
- black, non-transparent Eppis will protect compounds from light, but handling of small volumes of stock solutions is uncomfortable because it is hard to see the liquid in the Eppi.
- if the solvent is EtOH, glass vials are used instead of Eppis, because EtOH reduces the chemical stability of Eppi material (see Chemical stability of Eppendorf Consumables).
- DMSO can be stored in Eppendorf tubes. They are made of PP (polypropylene) which is compatible with DMSO according to Sigma-Aldrich's Product Information Sheet.
- Solutions stored in fridge or freezer should be prewarmed to room temperature before opening to avoid air-water condensation.
- Solutions should be mixed carefully to avoid phase separation that may occur in cold solutions, causing compounds’ precipitation.
- During the experiment, the stock solutions should be kept on ice.
- Some of the chemicals (e.g., ADP, NADH) are forbidden to be refrozen and new frozen aliquots are recommended to be used in each experiment.
- Some of the chemicals used have severe toxic effects on the environment. Make sure to dispose them correctly.
MitoPedia concepts: SUIT concept