MiPNet21.06 SUIT RP: Difference between revisions

Revision as of 19:26, 23 January 2016

SUIT reference assay for OXPHOS analysis by high-resolution respirometry. - »Bioblast pdf«

» Versions

OROBOROS (2016-01-23) Mitochondr Physiol Network

Abstract: Doerrier C, Sumbalova Z, Lamberti G, Krumschnabel G, Gnaiger (2016) SUIT reference assay for OXPHOS analysis by high-resolution respirometry. Mitochondr Physiol Network 21.06(pre03):1-10.

» Instrument: O2k, O2k-Catalogue

• O2k-Network Lab: AT_Innsbruck_OROBOROS

Labels: MiParea: Instruments;methods

Preparation: Permeabilized cells, Permeabilized tissue, Homogenate, Isolated mitochondria

Regulation: Substrate Coupling state: LEAK, OXPHOS, ETS"ETS" is not in the list (LEAK, ROUTINE, OXPHOS, ET) of allowed values for the "Coupling states" property.

HRR: Oxygraph-2k, Protocol"Protocol" is not in the list (Oxygraph-2k, TIP2k, O2k-Fluorometer, pH, NO, TPP, Ca, O2k-Spectrophotometer, O2k-Manual, O2k-Protocol, ...) of allowed values for the "Instrument and method" property.

O2k-SOP

Work in progress

Cytochrome c test

In protocol RP2, the cytochrome c test might better be moved from a c-titration after pyruvate (P) to a titration after M2. Otherwise, there is a risk of underestimation of FAO in cases of a cytochrome c effect.

~ Gnaiger Erich 18:26, 23 January 2016 (CET)

Mark names

Even the highly abbreviated names of respiratory states have become too long for DatLab, where mark names are restricted to 8 digits. The visibility in DatLab of long mark names is restricted. The simplest solution for short mark names is the use of a numerical sequence, with the preceeding event name added for information.

~ Gnaiger Erich 16:50, 23 January 2016 (CET)

Laboratory sheets for SUIT protocols RP1 and RP2

Type	SUIT protocol	Sample	Abbr	pdf file	docx file	Last update
Titration sheet	RP1	Permeabilized fibres	Pfi	»SUIT_RP1-Pfi	»SUIT_RP1-Pfi	2016-01-23
Titration sheet	RP2		Pfi	»SUIT_RP2-Pfi	»SUIT_RP2-Pfi	2016-01-23
Tick boxes	Combined		Pfi	»RP print-Pfi	»RP print-Pfi
Titration sheet	RP1	Permeabilized cells	Pc	»SUIT_RP1	»SUIT_RP1	2016-01-23
Titration sheet	RP2		Pc	»SUIT_RP2	»SUIT_RP2	2016-01-23
Tick boxes	Combined		Pc	»RP-Pc print	»RP-Pc print
Titration sheet	RP1	Isolated mitochondria, tissue homogenate	Imt, Thom	»SUIT_RP1	»SUIT_RP1
Titration sheet	RP2		Imt, Thom	»SUIT_RP2	»SUIT_RP2
Tick boxes	Combined		Imt, Thom	»RP print	»RP print

Experimental details

Work in progress towards the next pre-publication version of »MiPNet21.06 SUIT reference assay.pdf«

Saturating ADP concentrations

Different concentrations of ADP are sufficient to obtain maximum flux for estimating OXPHOS capacity. The typical range for isolated mitochondria is 1 to 2.5 mM, for permeabilized cells 1 to 5 mM, for permeabilized muscle fibres 2.5 to 10 mM.

Pre-publication communication

This communication is a pre-publication, inviting critical feedback and comments. All feedback will be carefully documented and evaluated in terms of justification of co-authorship. We intend to finally publish this topic in a peer-reviewed journal as an original article, with reference to the pre-publication history including reviewer's reports. - Gnaiger Erich 03:23, 18 January 2016 (CET)

Experiments in progress

The SUIT reference assay is presently applied in permeabilized HEK cells, mouse heart isolated mitochondria, liver homogenate, permeabilized skeletal muscle (mouse and human), and human PBMCs and platelets. - Gnaiger Erich 18:47, 19 January 2016 (CET)

AT Innsbruck Gnaiger E and AT Innsbruck OROBOROS - MitoFit project.
US NC Winston-Salem Molina AJA - UPBEAT project.

Further details

Introduction » Gnaiger 2014 MitoPathways

» MitoPedia: Respiratory substrate-coupling states - extended MitoPedia topic, Jan 2016

Table of titrations » MiPNet09.12 O2k-Titrations
Definition » Substrate-uncoupler-inhibitor titration
Context » SUIT protocol library
Abbreviations » MitoPedia

@@ Line 3: / Line 3: @@
 |info=[http://www.bioblast.at/index.php/File:MiPNet21.06_SUIT_reference_assay.pdf Versions]
 |authors=OROBOROS
-|year=2016-01-19
+|year=2016-01-23
 |journal=Mitochondr Physiol Network
 |abstract='''Doerrier C, Sumbalova Z, Lamberti G, Krumschnabel G, Gnaiger (2016) SUIT reference assay for OXPHOS analysis by high-resolution respirometry. Mitochondr Physiol Network 21.06(pre03):1-10.'''
@@ Line 19: / Line 19: @@
 }}
 ==  Work in progress ==
+=== Cytochrome c test ===
+* In protocol RP2, the cytochrome ''c'' test might better be moved from a c-titration after pyruvate (P) to a titration after M2. Otherwise, there is a risk of underestimation of FAO in cases of a cytochrome c effect.
+:~ [[User:Gnaiger Erich|Gnaiger Erich]] 18:26, 23 January 2016 (CET)
 === Mark names ===
 * Even the highly abbreviated names of respiratory states have become too long for DatLab, where mark names are restricted to 8 digits. The visibility in DatLab of long mark names is restricted. The simplest solution for short mark names is the use of a numerical sequence, with the preceeding event name added for information.
 :~ [[User:Gnaiger Erich|Gnaiger Erich]] 16:50, 23 January 2016 (CET)
 == Laboratory sheets for SUIT protocols RP1 and RP2 ==
@@ Line 40: / Line 46: @@
 ! pdf file
 ! docx file
+! Last update
 |-
 | Titration sheet
@@ Line 47: / Line 54: @@
 | [[Media:SUIT_RP1-Pfi.pdf |»SUIT_RP1-Pfi]]
 | [[Media:SUIT_RP1-Pfi.docx |»SUIT_RP1-Pfi]]
+| 2016-01-23
 |-
 | Titration sheet
@@ Line 54: / Line 62: @@
 | [[Media:SUIT_RP2-Pfi.pdf |»SUIT_RP2-Pfi]]
 | [[Media:SUIT_RP2-Pfi.docx |»SUIT_RP2-Pfi]]
+| 2016-01-23
 |-
 | Tick boxes
@@ Line 68: / Line 77: @@
 | [[Media:SUIT_RP1-Pc.pdf |»SUIT_RP1]]
 | [[Media:SUIT_RP1-Pc.docx |»SUIT_RP1]]
+| 2016-01-23
 |-
 | Titration sheet
@@ Line 75: / Line 85: @@
 | [[Media:SUIT_RP2-Pc.pdf |»SUIT_RP2]]
 | [[Media:SUIT_RP2-Pc.docx |»SUIT_RP2]]
+| 2016-01-23
 |-
 | Tick boxes