From Bioblast
- high-resolution terminology - matching measurements at high-resolution
Sample
Description
A sample is one or more parts taken from an ensemble that is studied. A sample is either stored for later quantification or prepared and possibly separated into subsamples, which are enclosed in a system for qualitative or quantitative investigation. A pure sample S is a pure gas, pure liquid or pure solid of a defined elementary entity-type. A pure biological sample is a cell type, tissue, or organism without its solid, liquid or gaseous environment. Then the system used to investigate sample S contains only entities of entity-type S, and the volume VS [L] and mass mS [kg] of the pure (sub)sample S are identical to the volume V and mass m of the experimental system. A pure sample S may be mixed with other components to be investigated as a solution, mixture, or suspension, indicated by the symbol s in contrast to the pure sample S. A sample s is obtained in combination with other components, such that the volume Vs [L] and mass ms [kg] of the sample s are larger than the volume VS and mass mS of the pure sample S. For example, the number of cells Nce [Mx] can be counted in a sample s of a cell suspension, whereas the mass mce [mg] of cells requires a pure sample S of cells to be measured on a mass-balance. Clarity of statistical representation is improved, if the symbol N is used for the number of primary samples taken from a study group, and the symbol n is used for the number of subsamples studied as technical repeats.
Abbreviation: s
Reference: BEC 2020.1
Communicated by Gnaiger Erich, last update 2024-12-05 by Cardoso Luiza and Timón-Gómez Alba
Sample type, sample and subsample
- A sample of a defined sample type subjected to a specific sample preparation (see MitoPedia: Sample preparations) may be, e.g., a mitochondrial preparation obtained from an organ, cells suspended from a cell culture dish, a tissue biopsy, an organ, an individual organism, or a group of organisms (e.g., a number of nematodes studied collectively in an experimental chamber).
- Example: Sample and subsample
- A blood sample or a biopsy taken from an individual organism. The sample size, N, corresponds to the number of replica, e.g., Norg organisms in a study group (arm). A sample may be processed and split into a number of subsamples (e.g., smaller volumes of blood taken from a larger volume of blood) for (i) application of different types of assay, and (ii) a number of repetitions or technical repeats, n, of the same assay, using subsamples of the same sample.
ISO 15189:2012
- A sample is one or more parts taken from a system and intended to provide information on the system, often to serve as a basis for evaluation of the system (diagnosis) or for a decision on intervention (therapy, production process) (ISO 15189:2012 Medical laboratories — Particular requirements for quality and competence).
- The term 'system' is used in this context with reference to a 'study system', distinguished from the 'system' in the context of thermodynamics (open, closed, isolated systems; instrumental chamber).
- Compare: Primary sample.
Application in HRR
Sample concentration
- The amount of tissue or cells used for high-resolution respirometry strongly depends on the sample characteristics and preparation.
- The sample concentration should be high enough to obtain a reliable respiratory flux per chamber volume, particularly when mitochondria have low respiratory activity. On the other hand, if volume-specific respiratory flux is too high, re-oxygenations have to be performed frequently disturbing the experimental course. As a general guideline:
- Maximum flux up to 100 to 300 pmol·s-1·mL-1; this is the most practical, yet far higher flux can be measured accurately with the Oroboros O2k and NextGen-O2k.
- Minimum flux at 5 pmol·s-1·mL-1 for the state with the lowest flux that will be analyzed, for instance, LEAK state.
- For permeabilized fibers, usually 0.7 mg tissue is used; for many cell types, often 1 million cells per mL can be used, however for some cell types it can be less or more depending on cell size and mitochondrial density. For PBMCs and Platelets, we recommend adding for PBMC 2·106 x/mL; for PLT 100·106 x/mL. Here are some examples of sample concentrations previously used with HRR:
- Isolated mitochondria:
- Mouse heart isolated mitochondria: 0.013−0.05 mg protein/mL (Cardoso 2021 BEC MgG, Leo 2024 MitoFit)
- Mouse brain isolated mitochondria: 0.09 mg protein/mL (Komlodi 2021 BEC Q)
- Tissue homogenates:
- Liver homogenate: 0.5 mg tissue wet mass/mL rat liver (Fischer 2022 Metabolites), 1 mg tissue wet mass/mL human liver biopsies (Meszaros 2024 Transpl Int)
- Brain homogenate: 1 mg tissue wet mass/mL mouse brain (Burtscher 2015 Mitochondrion, 1 mg tissue wet mass/mL mouse or rat brain (Cardoso 2025 Biochim Biophys Acta Mol Basis Dis)
- Kidney homogenate: 1 mg tissue wet mass/mL mouse kidney (Cardoso 2025 Biochim Biophys Acta Mol Basis Dis)
- Permeabilized tissue:
- Human skeletal muscle permeabilized fibers (vastus lateralis): 1.5 mg tissue wet mass or 0.04 mg mitochondrial protein in a 2-mL chamber (Boushel 2007 Diabetologia), 0.60–2.83 mg in a 2-mL chamber Doerrier 2024 Free Radic Biol Med
- Mouse heart permeabilized muscle fibers: 0.7–1.3 mg tissue wet mass in a 2 mL chamber (Lemieux 2017 Sci Rep)
- Biopsies: from 2 mg wet weight prostate tissue, punch needle biopsy, mechanically permeabilized (Schoepf 2016 FEBS J)
- Cells:
- HEK 293T cells: 1·106 cells/mL (Doerrier 2018 Methods Mol Biol, Baglivo 2024 MitoFit, Timon-Gomez 2024 MitoFit)
- Fibroblasts: 0.75·106 cells/mL human skin fibroblast cell lines (Leo 2024 MitoFit, Zdrazilova 2022 PLOS ONE), 0.2·106 cells/mL senescent human primary fibroblasts (Huetter 2004 Biochem_J)
- PBMCs: 5–6·106 cells/mL human PBMCs (Timon-Gomez 2024 MitoFit), 2·106 cells/mL rat PBMCs (Fischer 2022 Metabolites)
- Human platelets: 100–260·106 platelets/mL (Timon-Gomez 2024 MitoFit, Leo 2024 MitoFit, Palacka 2021 Int J Mol Sci, Vernerova 2021 Biomedicines, Sumbalova 2022 Front_Mol_Biosci)
- Droshophila melanogaster:
- 0.05 mg/mL thorax isolated mitochondria (Rodriguez 2023 BEC)
- 10–12 Droshophila brains per 2-mL chamber (Cardoso 2025 Biochim Biophys Acta Mol Basis Dis)
- Algae:
- Nannochloropsis gaditana, strain CCAP 849/5: 5–10·106 cells (Vera-Vives 2022 BEC)
- For permeabilized fibers, usually 0.7 mg tissue is used; for many cell types, often 1 million cells per mL can be used, however for some cell types it can be less or more depending on cell size and mitochondrial density. For PBMCs and Platelets, we recommend adding for PBMC 2·106 x/mL; for PLT 100·106 x/mL. Here are some examples of sample concentrations previously used with HRR:
- For more information about living cells, see: Appropriate cell-count concentration for HRR.
- For other sample types, it might be possible to find previous publications using the Oroboros with the same sample or similar ones, to have an estimate of the necessary sample amount. See: O2k-Publications: Organisms and O2k-Publications: Tissues and cells
- Of note, fluorometric applications may require a higher sample amount than necessary for high-resolution respirometry.
O2k-sV-Module
- With the O2k-sV-Module (included in all O2k-packages), the amount of cells or tissue required can be decreased by a factor of 4 in comparison to the standard chambers (2 mL) - Leo_2024_MitoFit
Addition of sample to the respirometer chamber
- Isolated mitochondria (imt): imt are typically added with a Hamilton syringe. Care should be taken to not exert damaging low pressure during aspiration of the sample into the syringe or exerting high pressure during injection into the chamber.
- Living cells (ce) are typically added with a pipette because some cell types may be easily damaged when injected with a syringe. Care should be taken to not exert damaging low pressure during aspiration of the sample into the pipette or exerting high pressure during injection into the chamber. We recommend removing the stopper, replacing a defined volume of the respiration medium with cell suspension, and closing the chamber again. Sometimes (i.e. microalgal cells) the whole volume of the chamber (2 mL) is replaced by the cells suspension with the cell concentration adjusted to the desired value. Further details: »Cell count and normalization in HRR.
- Permeabilized tissue (pti)/Permeabilized muscle fibers (pfi): Remove the Stopper, add the permeabilized tissue/fibers into the chamber, close the chamber again.
- Permeabilized cells (pce): Similar to living cells, permeabilized cells are added using a pipette. Remove the Stopper, replace a defined volume (partial or total) of the respiration medium with cell suspension, close the chamber again.
For more information:
Addition of biological sample to the respirometer chamber
Fragile samples
- The rigorous stirring required for high-resolution respirometry causes problems for organisms / samples that are easily damaged by mechanical force, such as fish eggs, brain slices, or beads used to grow adherent cells.
Sample holder
- The Sample Holder was developed to separate the sample from the stirrer compartment by a stainless steel mesh, without interfering with the gentle mixing of the aqueous solution between the two compartments. Samples must be large enough not to pass through the 0.1 mm pore size of the mesh.
- Sample Holder - manual and videosupport
- Dias C, Lourenco CF, Barbosa RM, Laranjinha J, Ledo A (2018) Analysis of respiratory capacity in brain tissue preparations: high-resolution respirometry for intact hippocampal slices - »Bioblast link« - Original publication on the tissue holder prototype with brain slices.
- Dias C, Lourenço CF, Laranjinha J, Ledo A (2022) Modulation of oxidative neurometabolism in ischemia/reperfusion by nitrite - »Bioblast link« - Tissue holder prototype with brain slices.
- The Sample Holder was developed to separate the sample from the stirrer compartment by a stainless steel mesh, without interfering with the gentle mixing of the aqueous solution between the two compartments. Samples must be large enough not to pass through the 0.1 mm pore size of the mesh.
Smaller stirring bars?
- Question: Would smaller stirring bars help to minimize damage (combined with lower stirring speed)? Would they be available?
- Answer: we are not sure if smaller stirring bars would help to avoid damage of fragile samples. In the early phase of the development of the Oroboros many different geometries and sizes of stirring bars were tested. In any case, commercially available Teflon (R) coated stirring bars cannot be used for high-resolution respirometry due to the high oxygen solubility of Teflon and the corresponding unacceptably high O2 background flux. There is the possibility of reducing the stirring speed. The minimum practical stirring speed is 550 rpm, to avoid that the noise of the O2 slope exceeds the threshold of ±4 pmol∙s-1∙mL-1 (Leo 2024 MitoFit).
References
| Bioblast link | Reference | Year |
|---|---|---|
| Gnaiger 2020 BEC MitoPathways | Gnaiger E (2020) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 5th ed. Bioenerg Commun 2020.2. https://doi.org/10.26124/bec:2020-0002 | 2020 |
| BEC 2020.1 doi10.26124bec2020-0001.v1 | Gnaiger E et al ― MitoEAGLE Task Group (2020) Mitochondrial physiology. Bioenerg Commun 2020.1. https://doi.org/10.26124/bec:2020-0001.v1 | 2020 |
MitoPedia O2k and high-resolution respirometry: Oroboros QM
MitoPedia topics:
Sample preparation
