Difference between revisions of "MitoEAGLE blood cells 1"
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|year=2018 | |year=2018 | ||
|journal=MitoEAGLE preprint | |journal=MitoEAGLE preprint | ||
|abstract= | |abstract=::: '''Version beta 01''' | ||
:::::# The evaluation of mitochondrial function remains crucial for the diagnosis of a spectrum of human pathologies. Peripheral blood provides an easily accessible source of biological material like cells. However, mitochondrial respiratory studies in blood cells still require standardization. The procedures applied in bioenergetic assessments involve multiple steps including pre-analytical analytical and normalization phase. Therefore, the aim of this report is to review and summarize methods using peripheral blood mononuclear cells and platelets for mitochondrial respiratory studies. For this purpose we have used original data from multiple laboratories combined with literature data. | |||
:::::# This methodological review is not primarily to present results on patients, but to characterize control groups for pathophysiological studies. During pre-analytical phase smoking, alcohol intake, BMI threshold, lifestyle intervention and medications should be considered for inclusion / exclusion of controls. Moreover, all subjects should be matched for sex, age and ethnicity. Conditions like time of sampling, fasting and resting state are terms of standardization. | |||
:::::# Anticoagulants are used for whole blood sampling /e.g. K-EDTA, lithium heparin, sodium citrate/ and discussed for their effect on cells isolation, yield, cells fraction purity and respiration. | |||
:::::# The time and temperature during transport and storage of whole blood before specific cells separation influences next analytical phases and affect final outcome. Characterization of whole blood provides the basis for inclusion / exclusion for further processing of the sample. | |||
:::::# Separation procedures depend on the type of cells required for respirometric studies / PLT, PBMC or PLT and PBMC/. Media used, centrifugation conditions, cell counting and cell viability methods are under discussion. Purity of preparation / e.g. PLT contamination in PBMCs fraction, purity of PLT fraction/, recovery and yield of cells from the whole blood are also presented in this report. | |||
:::::# There are limited data on the effect of storage and conditions during processing of isolated cell types before respirometry. Temperature, media, antibiotics, proteinase inhibitors cocktail, density of cells during storage, tilting of cell fraction might significantly affect the measurements. | |||
:::::# Specific protocols are used for respiration assessments in intact and permeabilized. The type of medium used for respirometry /MiR 05, RPMI, plasma/ need further evaluations. | |||
:::::# Finally, we focus on the normalization, which is important to interpret correctly and compare the results of respiratory studies. It should include data on contamination of PBMCs with PLTs, cell viability /e.g. succinate test/, flux control ratios, total protein concentration, citrate synthase activity. Some other markers, like clusters of differentiation specific for leukocytes and platelets can be taken into consideration. | |||
:::::# To conclude, we summarize emerging recommendations for mitochondrial respiratory studies in intact and permeabilized PBMCs and PLTs towards building a data base of healthy subjects that can be used as controls in clinical studies. | |||
}} | }} | ||
{{Labeling | {{Labeling | ||
|area=Respiration, Instruments;methods, mtDNA;mt-genetics, nDNA;cell genetics, Gender | |area=Respiration, Instruments;methods, mtDNA;mt-genetics, nDNA;cell genetics, Gender | ||
| Line 21: | Line 26: | ||
|pathways=F, N, S, Gp, CIV, NS, Other combinations, ROX | |pathways=F, N, S, Gp, CIV, NS, Other combinations, ROX | ||
}} | }} | ||
:::: Co-authors: The present alphabetical list of co-authors icludes all actively involved participants of [[MitoEAGLE Innsbruck 2018]] and will be extended by further contributors at [[MitoEAGLE Poznan 2018]] and by MitoEAGLE members submitting their valuable contributions. All co-authors will have to confirm to have made a contribution and to have read the final manuscript. | |||
<br/> | |||
[[File:Workflow.png|500px|left|thumb|Fig. 1. Workflow and topics]] | |||
[[Talk:MitoEAGLE blood cells 1 |''Work in progress'']] - Innsbruck retreat for preparation of '''[[MitoEAGLE Poznan 2018]]''' | |||
» [[WG4_MitoEAGLE_data:_blood_and_cultured_cells#TG4.1_Blood |MitoEAGLE blood cells group (WG 4)]] | |||
Revision as of 16:07, 6 February 2018
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COST Action CA15203 (2016-2021): MitoEAGLE
Evolution-Age-Gender-Lifestyle-Environment: mitochondrial fitness mapping
MitoEAGLE blood cells 1
| MitoEAGLE blood cells group 2018-01-29 An interlaboratory guide through procedures for mitochondrial respiratory studies with intact and permeabilized peripheral blood mononuclear cells and platelets. |
Calabria E, Chang SC, Duicu OM, Garcia-Souza LF, Gnaiger E, Labieniec-Watala M, Michalak S, Siewiera K, Silaidos C, Sumbalova Z, Volani C (2018) MitoEAGLE preprint
Abstract:
- Version beta 01
- The evaluation of mitochondrial function remains crucial for the diagnosis of a spectrum of human pathologies. Peripheral blood provides an easily accessible source of biological material like cells. However, mitochondrial respiratory studies in blood cells still require standardization. The procedures applied in bioenergetic assessments involve multiple steps including pre-analytical analytical and normalization phase. Therefore, the aim of this report is to review and summarize methods using peripheral blood mononuclear cells and platelets for mitochondrial respiratory studies. For this purpose we have used original data from multiple laboratories combined with literature data.
- This methodological review is not primarily to present results on patients, but to characterize control groups for pathophysiological studies. During pre-analytical phase smoking, alcohol intake, BMI threshold, lifestyle intervention and medications should be considered for inclusion / exclusion of controls. Moreover, all subjects should be matched for sex, age and ethnicity. Conditions like time of sampling, fasting and resting state are terms of standardization.
- Anticoagulants are used for whole blood sampling /e.g. K-EDTA, lithium heparin, sodium citrate/ and discussed for their effect on cells isolation, yield, cells fraction purity and respiration.
- The time and temperature during transport and storage of whole blood before specific cells separation influences next analytical phases and affect final outcome. Characterization of whole blood provides the basis for inclusion / exclusion for further processing of the sample.
- Separation procedures depend on the type of cells required for respirometric studies / PLT, PBMC or PLT and PBMC/. Media used, centrifugation conditions, cell counting and cell viability methods are under discussion. Purity of preparation / e.g. PLT contamination in PBMCs fraction, purity of PLT fraction/, recovery and yield of cells from the whole blood are also presented in this report.
- There are limited data on the effect of storage and conditions during processing of isolated cell types before respirometry. Temperature, media, antibiotics, proteinase inhibitors cocktail, density of cells during storage, tilting of cell fraction might significantly affect the measurements.
- Specific protocols are used for respiration assessments in intact and permeabilized. The type of medium used for respirometry /MiR 05, RPMI, plasma/ need further evaluations.
- Finally, we focus on the normalization, which is important to interpret correctly and compare the results of respiratory studies. It should include data on contamination of PBMCs with PLTs, cell viability /e.g. succinate test/, flux control ratios, total protein concentration, citrate synthase activity. Some other markers, like clusters of differentiation specific for leukocytes and platelets can be taken into consideration.
- To conclude, we summarize emerging recommendations for mitochondrial respiratory studies in intact and permeabilized PBMCs and PLTs towards building a data base of healthy subjects that can be used as controls in clinical studies.
- Version beta 01
Labels: MiParea: Respiration, Instruments;methods, mtDNA;mt-genetics, nDNA;cell genetics, Gender
Pathology: Aging;senescence
Organism: Human Tissue;cell: Blood cells, Platelet Preparation: Intact cells, Permeabilized cells Enzyme: Marker enzyme
Coupling state: LEAK, ROUTINE, OXPHOS, ET Pathway: F, N, S, Gp, CIV, NS, Other combinations, ROX
- Co-authors: The present alphabetical list of co-authors icludes all actively involved participants of MitoEAGLE Innsbruck 2018 and will be extended by further contributors at MitoEAGLE Poznan 2018 and by MitoEAGLE members submitting their valuable contributions. All co-authors will have to confirm to have made a contribution and to have read the final manuscript.
File:Workflow.png
Fig. 1. Workflow and topics
Work in progress - Innsbruck retreat for preparation of MitoEAGLE Poznan 2018
