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Excel templ: Protocols

From Bioblast

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Disclaimer: This is an alpha version and page is a currently still under development. Please DO NOT consider information below in this page to be true or of any scientific value.

This page was created to provide a list and details about DatLab protocols for analysis of mitochondrial respiration.

Data for Excel template

Protocol name DLP# Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Step 7 Step 8 Step 9 Step 10 Step 11 Step 12 Step 13 Step 14 Step 15 Step 16 Step 17 Step 18 Step 19 Step 20 # of steps State 1 State 2 State 3 State 4 State 5 State 6 State 7 State 8 State 9 State 10 State 11 State 12 State 13 State 14 State 15 State 16 State 17 State 18 State 19 State 20 Reference step Reference Mark Baseline step Baseline Mark Short name Notes Descriptions
SUIT-001_O2_mt_D001 D001 1mt 1PM 2D 2c 3U 4G 5S 6Oct 7Rot 8Gp 9Ama 10AsTm 11Azd 13 ROX N_L N_P N_P N_E N_E NS_E FNS_E S_E SGp_E ROX CIV ROX 7 5S 11 9Ama RP1_mt Doerrier 2018 Methods Mol Biol The SUIT-001 O2 mt D001 protocol in combination with SUIT-002 O2 mt D005 is specially designed to provide a common reference for comparison of respiratory control of mitochondrial preparations such as isolated mitochondria, tissue homogenates and permeabilized cells (already permeabilized when they are added to the chamber) in a wide variety of species and tissues. SUIT-001 O2 mt D001 gives information of the linear coupling control (L- P- E) with NADH linked-substrates (PM). Moreover, the pathway control in ET state (N, NS, FNS, S and SGp pathways) can be evaluated by using this SUIT protocol. SUIT-001 O2 mt D001 can be extended with the CIV assay module.
SUIT-001_O2_pfi_D002 D002 1pfi 1PM 2D 2c 3U 4G 5S 6Oct 7Rot 8Gp 9Ama 10AsTm 11Azd 13 ROX N_L N_P N_P N_E N_E NS_E FNS_E S_E SGp_E ROX CIV ROX 7 5S 11 9Ama RP1_pfi Doerrier 2018 Methods Mol Biol The SUIT-001 O2 pfi D002 protocol in combination with SUIT-002_O2_pfi_D006 is specially designed to provide a common reference for comparison of respiratory control of permeabilized fibers in a wide variety of species and tissues. SUIT-001 O2 pfi D002 gives information of the linear coupling control (L- P- E) with NADH linked-substrates (PM). Moreover, the pathway control in ET state (N, NS, FNS, S and SGp pathways) can be evaluated by using this SUIT protocol. SUIT-001 O2 pfi D002 can be extended with the CIV assay module.
SUIT-001_O2_ce-pce_D003 D003 ce1 1Dig 1PM 2D 2c 3U 4G 5S 6Oct 7Rot 8Gp 9Ama 10AsTm 11Azd 14 ce_R ROX N_L N_P N_P N_E N_E NS_E FNS_E S_E SGp_E ROX CIV ROX 8 5S 12 9Ama RP1_pce Doerrier 2018 Methods Mol Biol The SUIT-001 O2 ce-pce D003 protocol in combination with SUIT-002 O2 ce-pce D007 is specially designed to provide a common reference for comparison of respiratory control of permeabilized cells (non-permeabilized cells must be added to the chambers and then permeabilized as a part of the protocol) in a wide variety of cell types. SUIT-001 O2 ce-pce D003 gives information on ROUTINE respiration (ce: non-permeabilized cells) and after the plasma membrane permeabilization (pce: permeabilized cells) on linear coupling control (L- P- E) with NADH linked-substrates (PM). Moreover, the pathway control in ET state (N, NS, FNS, S and SGp pathways) can be evaluated by using this SUIT protocol. SUIT-001 O2 ce-pce D003 can be extended with the CIV assay module.
SUIT-001_O2_PBMC-PLT_D004 D004 ce1 1Dig 1PM 2D 2c 3U 4G 5S (6Oct) 7Rot 8Gp 9Ama 10AsTm 11Azd 14 ce_R ROX N_L N_P N_P N_E N_E NS_E S_E SGp_E ROX CIV ROX 8 5S 12 9Ama RP1_PBMC-PLT Doerrier 2018 Methods Mol Biol The SUIT-001 O2 ce-pce D004 protocol is the reference protocol 1 (RP1) specifically for PBMCs (Peripheral Blood Mononuclear Cells) and PLTs (platelets). The main difference between the RP1 for PBMCs and PLTs and RP1 for other mitochondrial preparations (see SUIT-001) is the absence of Oct in the case of blood cells.

The SUIT-001 O2 ce-pce D004 protocol in combination with SUIT-002 O2 ce-pce D007a is specially designed to provide a common reference for comparison of respiratory control of PBMC and platelets cells (non-permeabilized cells must be added to the chambers and then permeabilized as a part of the protocol) in a wide variety of species. SUIT-001 O2 ce-pce D004 gives information of ROUTINE respiration (ce: non-permeabilized cells) and after the plasma membrane permeabilization (pce: permeabilized cells) of the linear coupling control (L- P- E) with NADH linked-substrates (PM). Moreover, the pathway control in ET state (N, NS, S and SGp pathways) can be evaluated by using this SUIT protocol. SUIT-001 O2 ce-pce D004 can be extended with the CIV assay module.

SUIT-002_O2_mt_D005 D005 1mt 1D 2M.1 3Oct 3c 4M2 5P 6G 7S 8Gp 9U 10Rot 11Ama 12AsTm 13Azd 15 ROX ROX F_P F_P F(N)_P FN_P FN_P FNS_P FNSGp_P FNSGp_E SGp_E ROX CIV ROX 11 9U 13 11Ama RP2_mt A: Doerrier 2018 Methods Mol Biol The SUIT-002 O2 mt D005 protocol in combination with SUIT-001_O2_mt_D001 provides a common reference for comparison of respiratory control of mitochondrial preparations such as isolated mitochondria, tissue homogenates and permeabilized cells (already permeabilized when they are added to the chamber) in a wide variety of species, tissues and cell types. SUIT-002 O2 mt D005 is specially designed to give information on F-pathway in OXPHOS state avoiding FAO overestimation in the presence of anaplerotic pathways. Moreover, the pathway control in OXPHOS state (F, F(N), FN, FNS, FNSGp pathways) and in ET state (FNSGp and SGp) can be evaluated by using this SUIT protocol. SUIT-002 O2 mt D005 can be extended with the CIV assay module.
SUIT-002_O2_pfi_D006 D006 1pfi 1D 2M.1 3Oct 3c 4M2 5P 6G 7S 8Gp 9U 10Rot 11Ama 12AsTm 13Azd 15 ROX ROX F_P F_P F(N)_P FN_P FN_P FNS_P FNSGp_P FNSGp_E SGp_E ROX CIV ROX 11 9U 13 11Ama RP2_pfi A: Doerrier 2018 Methods Mol Biol The SUIT-002 pfi D006 protocol in combination with SUIT-001_O2_pfi_D002 provides a common reference for comparison of respiratory control of permeabilized muscle fibers in a wide variety of species and tissues. SUIT-002 pfi D006 is specially designed to give information on F-pathway in OXPHOS state avoiding FAO overestimation in the presence of anaplerotic pathways. Moreover, the pathway control in OXPHOS state (F, F(N), FN, FNS, FNSGp pathways) and in ET state (FNSGp and SGp) can be evaluated by using this SUIT protocol. SUIT-002 pfi D006 can be extended with the CIV assay module.
SUIT-002_O2_ce-pce_D007 D007 ce1 1Dig 1D 2M.1 3Oct 3c 4M2 5P 6G 7S 8Gp 9U 10Rot 11Ama 12AsTm 13Azd 16 ce_R ROX ROX F_P F_P F(N)_P FN_P FN_P FNS_P FNSGp_P FNSGp_E SGp_E ROX CIV ROX 12 9U 14 11Ama RP2_pce A: Doerrier 2018 Methods Mol Biol The SUIT-002 O2 ce-pce D007 protocol in combination with SUIT-001 O2 ce-pce D003 provides a common reference for comparison of respiratory control of permeabilized cells (non-permeabilized cells must be added to the chambers and then permeabilized as a part of the protocol) in a wide variety of cell types. SUIT-002 O2 ce-pce D007 is specially designed to give information on F-pathway in OXPHOS state avoiding FAO overestimation in the presence of anaplerotic pathways. Moreover, the pathway control in OXPHOS state (F, F(N), FN, FNS, FNSGp pathways) and in ET state (FNSGp and SGp) can be evaluated by using this SUIT protocol. SUIT-002 O2 ce-pce D007 can be extended with the CIV assay module.
SUIT-002_O2_ce-pce-PBMC-PLT_D007a D007a ce1 1Dig 1D 2M.1 3Oct 3c 4M2 5P 6G 7S 8Gp 9U 10Rot 11Ama 12AsTm 13Azd 16 ce_R ROX ROX F_P F_P F(N)_P FN_P FN_P FNS_P FNSGp_P FNSGp_E SGp_E ROX CIV ROX 12 9U 14 11Ama RP2_PBMC-PLT A: Doerrier 2018 Methods Mol Biol The SUIT-002 O2 ce-pce D007a protocol is the reference protocol 2 (RP2) specifically for PBMCs and PLTs. The main difference between the RP2 for PBMCs and PLTs and RP2 for other permeabilized cells (SUIT-002 O2 ce-pce D007) is the volume/concentration of the titrations.

The SUIT-002 O2 ce-pce D007a protocol in combination with SUIT-001 O2 ce-pce D004 provides a common reference for comparison of respiratory control of PBMC and platelets cells in a wide variety of species. SUIT-002 O2 ce-pce D007a is specially designed to give information on F-pathway in OXPHOS state avoiding FAO overestimation in the presence of anaplerotic pathways. Moreover, the pathway control in OXPHOS state (F, F(N), FN, FNS, FNSGp pathways) and in ET state (FNSGp and SGp) can be evaluated by using this SUIT protocol. SUIT-002 O2 ce-pce D007a can be extended with the CIV assay module.

SUIT-010_O2_ce-pce_D008 D008 ce1 ce2Rot ce3S ce4D 1Dig 1c 6 ce_R ROX ceS_L ceS_P S_P S_P 5 1Dig 2 ce2Rot Digitonin test A: Optimization of digitonin concentration for pce SUIT-010 O2 ce-pce D008 is designed for the evaluation of optimum digitonin concentration for selective cell membrane permeabilization, a requirement to account for differences between cell types, the concentration of cells, and variability between batches of digitonin. After inhibition of the endogenous ROUTINE respiration by rotenone, respiration of intact cells is not stimulated by succinate and ADP. Subsequent stepwise digitonin titration yields gradual permeabilization of the cell membrane, indicating by the increase of respiration up to full permeabilization. Respiration is constant within optimal digitonin concentration and inhibited by over-titration with digitonin, since the mitochondrial outer membrane is affected and cytochrome c can be released. Therefore, it is recommended to titrate cytochrome c after the optimal digitonin concentration has been reached. In other SUIT protocols for permeabilized cells, the respiration of pce is stable, if the optimal digitonin concentration is used.
SUIT-003_O2_ce_D009 D009 ce1 ce2Omy ce3U ce4Ama 4 ce_R ce_L ce_E ROX 3 ce3U 4 ce4Ama CCP-ce A: Coupling control protocol, living cells »MiPNet08.09 CellRespiration Bioblast pdf The SUIT-003 O2 ce D009 (CCP-ce) is designed to study coupling control of living cells. Respiratory capacities are tested in a sequence of coupling states: ROUTINE, LEAK respiration and Electron transfer pathway. To study LEAK respiration, the phosphorylation system is inhibited by oligomycin. The final concentration of oligomycin has to be carefully optimized for various cell types, to minimize the inhibitory effect on the electron transfer system which would lead to an underestimation of ET capacity.
SUIT-004_O2_pfi_D010 D010 1pfi 1PM 2D 2c 3U 4S 5Rot 6Ama 7AsTm 8Azd 10 ROX N_L N_P N_P N_E NS_E S_E ROX CIV ROX 6 4S 8 6Ama RP1-short The SUIT-004 O2 pfi D010 protocol provides information of the linear coupling control (L- P- E) with NADH linked-substrates (PM). Moreover, the pathway control in ET state (N, NS and S) can be evaluated by using this SUIT protocol. The SUIT-004 O2 pfi D010 can be extended with the CIV assay module.
SUIT-005_O2_pfi_D011 D011 1pfi 1OctM 2D 2c 3P 4S 5U 6Rot 7Ama 8AsTm 9Azd 11 ROX F(N)_L F(N)_P F(N)_P FN_P FNS_P FNS_E S_E ROX CIV ROX 7 5U 9 7Ama RP2-short A: when malate-anaplerotic activity is zero SUIT-005 O2 pfi D011 protocol provides information on the F-pathway, the combined FN pathway, and the convergence FNS pathways in the OXPHOS state. FNS comprises the most important pathways in many cell types and thus provides a physiologically relevant estimate of maximum OXPHOS- and ET capacity. SUIT-005 can be extended with the CIV assay module.

This protocol is linked to SUIT-002 O2 pfi D006 - SUIT RP2, specifically for human skeletal muscle mitochondria. SUIT-005 O2 pfi D011 is harmonized with SUIT-004 O2 pfi D010. The F-pathway has to be tested previously.

SUIT-003_O2_ce_D012 D012 ce1 ce2P ce2Omy ce3U ce4Rot ce5Ama 6 ce_R ce_R ce_L ce_E ROX ROX 4 ce3U 6 ce5Ama CCP-ce_+P A: Coupling control protocol, living cells »MiPNet08.09 CellRespiration Bioblast pdf SUIT-003 O2 ce D012 is a coupling-control protocol for living cells. Different coupling control states are assessed (ROUTINE - L(Omy) - E) at ce-substrate state. The use of pyruvate is recommended to support the cell respiration if the incubation media is not cell culture media. The use of oligomycin provides important information about the ETS and TCA activity but the optimal concentration has to be carefully determined experimentally to avoid the known side effects over the ET-capacity. Rotenone and Antimycin A provide the ROX state and its sequential addition allows the extension of the protocol with the cell viability test module (See CCV SUIT-003 O2 ce-pce D020).
SUIT-003_O2_ce-pce_D013 D013 ce1 ce1P ce2Omy ce3U ce3Glc ce3M ce4Rot ce5S 1Dig 1U 1c 2Ama 3AsTm 4Azd 14 ce_R ce_R ce_L ce_E ce_E ce_E ROX ceS_E S_E S_E S_E ROX CIV ROX 4 ce3U 12 2Ama CCP-ce_+Glc+M+viability+CIV A: Coupling control protocol, living cells »MiPNet08.09 CellRespiration Bioblast pdf SUIT-003 O2 ce-pce D013 is designed to study coupling control and plasma membrane permeability of living cells. Respiratory capacities are tested in a sequence of coupling states: ROUTINE, LEAK respiration and Electron transfer pathway in the presence of glucose and malate. To study LEAK respiration, the phosphorylation system is inhibited by oligomycin. The final concentration of oligomycin has to be carefully optimized for various cell types, to minimize the inhibitory effect on the electron transfer system which would lead to an underestimation of ET capacity. At Electron transfer pathway state, glucose is added to test the Crabtree effect on living cells. Malate is added to stimulate respiration of cells without intact plasma membrane (dead cells, dce). The viability index, VI, in the simple CCVP-Glc,M (SUIT-003 O2 ce-pce D013) may be underestimated if succinate uptake in the living cells proceeds through the dicarboxylate carrier of the plasma membrane. CCVP-Glc,M can be extended by the Complex IV module.
SUIT-008_O2_pfi_D014 D014 1pfi 1PM 2D 2c 3G 4S 5U 6Rot 7Ama 8AsTm 9Azd 11 ROX N_L N_P N_P N_P NS_P NS_E S_E ROX CIV ROX 7 5U 9 7Ama PM+G+S_OXPHOS+Rot_ET_pfi A: Additivity between the N- and S-pathway in the Q-junction The SUIT-008 O2 pfi D014 protocol is designed to assess the additivity between the N- and S-pathway in the Q-junction, providing a physiologically relevant estimate of maximum mitochondrial respiratory capacity in permeabilized fibers. It also serves as a diagnostic tool for the activity of the glutamate dehydrogenase and its linked pathways, which could be relevant in some pathologies. SUIT-008 O2 pfi D014 can be easily extended with the CIV assay module.
SUIT-009_O2_mt_D015 D015 1mt 1S 2D 2c 3P 4Rot 5Ama 7 ROX S_L S_P S_P NS_P S_P ROX 5 3P 7 5Ama S_OXPHOS_Rot_O2_mt SUIT-009 O2 mt D015 is a short protocol for determination of O2 flux in isolated mitochondria, tissue homogenate and permeabilized cells (already permeabilized when they are added to the chamber). It is recommended as a control experiment for SUIT-009 AmR mt D021 without using the Amplex UltraRed assay in order to detect the effect of the fluorescent dye on the O2 flux and with the cytochrome c to assess the integrity of the mitochondrial outer membrane. We do not recommend this protocol for determination of the O2 flux in the absence of Rotenone. The following Coupling-control protocol can be used to assess Succinate-linked respiration with Rotenone: SUIT-006 O2 mt D022.
SUIT-009_O2_ce-pce_D016 D016 ce1 1Dig 1S 2D 2c 3P 4Rot 5Ama 8 ce_R ROX S_L S_P S_P NS_P S_P ROX 6 3P 8 5Ama S_OXPHOS_Rot_O2_pce SUIT-009 O2 ce-pce D016 is a short protocol to investigate O2 flux in permeabilized cells (non-permeabilized cells must be added to the chambers and then permeabilized as a part of the protocol). It is recommended as a control experiment for SUIT-009 AmR ce-pce D019 without using the Amplex UltraRed assay in order to detect the effect of the fluorescence dye on the O2 flux and with the cytochrome c to assess the integrity of the mitochondrial outer membrane. We do not recommend this protocol for determination of the O2 flux in the absence of Rotenone. The following Coupling-control protocol can be used to assess Succinate-linked respiration with Rotenone: SUIT-006 O2 mt D022.
SUIT-003_O2_ce-pce_D018 D018 ce1 ce1P ce2Omy ce3U ce3Glc ce4Rot ce5S 1Dig 1c 2Ama 3AsTm 4Azd 12 ce_R ce_R ce_L ce_E ce_E ROX ceS_E S_E S_E ROX CIV ROX 4 ce3U 10 2Ama CCP-ce_+Glc+viability+CIV A: Coupling control protocol, living cells »MiPNet08.09 CellRespiration Bioblast pdf SUIT-003 O2 ce-pce D018 is designed to study coupling control and plasma membrane permeability of living cells. Respiratory capacities are tested in a sequence of coupling states: ROUTINE, LEAK respiration and Electron transfer pathway in the presence of glucose. To study LEAK respiration, the phosphorylation system is inhibited by oligomycin. The final concentration of oligomycin has to be carefully optimized for various cell types, to minimize the inhibitory effect on the electron transfer system which would lead to an underestimation of ET capacity. At Electron transfer pathway state, glucose is added to test the Crabtree effect on living cells. The viability index, VI, in the simple CCVP-Glc (SUIT-003 O2 ce-pce D018) may be underestimated if succinate uptake in the living cells proceeds through the dicarboxylate carrier of the plasma membrane. CCVP-Glc can be extended by the Complex IV module.
SUIT-003_O2_ce-pce_D020 D020 ce1 ce1P ce2Omy ce3U ce4Rot ce5S 1Dig 1c 2Ama 3AsTm 4Azd 11 ce_R ce_R ce_L ce_E ROX ceS_E S_E S_E ROX CIV ROX 4 ce3U 9 2Ama CCP-ce_+viability+CIV A: Coupling control protocol, living cells »MiPNet08.09 CellRespiration Bioblast pdf SUIT-003 O2 ce-pce D020 is designed to study coupling control and plasma membrane permeability of living cells. Respiratory capacities are tested in a sequence of coupling states: ROUTINE, LEAK respiration and Electron transfer pathway. To study LEAK respiration, the phosphorylation system is inhibited by oligomycin. The final concentration of oligomycin has to be carefully optimized for various cell types, to minimize the inhibitory effect on the electron transfer system which would lead to an underestimation of ET capacity. The viability index, VI, in the simple CCVP-ce (SUIT-003 O2 ce-pce D020) may be underestimated, if succinate uptake in the living cells proceeds throught the dicarboxylate carrier of the plasma membrane. CCVP-ce can be extended by the Complex IV module.
SUIT-006_O2_mt_D022 D022 1mt 1S(Rot) 2D 2c (3Omy) 4U 5Ama 7 ROX S_L S_P S_P S_E ROX 6 4U 7 5Ama CCP-mtprep_Srot A: Coupling control protocol, mtprep SUIT-006 O2 mt D022 is a coupling-control protocol for mitochondrial preparations (tissue homogenate, isolated mitochondria and permeabilized cells). Different coupling control states are assessed (L(n) - P - E) at succinate pathway control state. The use of rotenone, that inhibits complex I, provides a specific coupling-control protocol for S-pathway, avoiding reverse electron transfer (RET).

NOTE: In the DatLab software, the DLP file for SUIT-006 O2 mt D022 is provided without the addition of oligomycin. For using this protocol with oligomycin addition, a DLPU can be created, with the Omy event and mark. Using oligomycin in the preliminary assays with a not yet studied model is useful to determine whether in this kind of sample LEAK respiration in the absence of ADP (1SRot) may be overestimated due to the presence of residual and endogenous adenylates.

SUIT-011_O2_pfi_D024 D024 1pfi 1GM 2D 2c 3S 4U 5Rot 6Ama 8 ROX N_L N_P N_P NS_P NS_E S_E ROX 6 4U 8 6Ama GM+S_OXPHOS+Rot_ET:pfi A: Maximum mitochondrial respiratory capacity (OXPHOS with NS substrates) and coupling/pathway control The SUIT-011 O2 pfi D024 protocol is designed to study physiologically relevant maximum mitochondrial respiratory capacity (OXPHOS with NS substrates) and coupling/pathway control states. SUIT-011 gives information of the linear coupling control (L- P) with NADH linked-substrates (GM). GM and PM yield practically identical fluxes in human skeletal muscle fibers. However, PM is the superior alternative to GM, since the fractions of the N-pathway is lower and of the S-pathway is higher with GM compared to PM. PM, therefore, yields a more sensitive assay for the diagnosis of injuries in the N-linked pathway (compare SUIT-001 and SUIT-004). Moreover, SUIT-011 allows the evaluation of the coupling control state (P- E) with NADH and succinate linked-substrates (NS) and the pathway control in OXPHOS (NS) and ET state (NS and S). SUIT-011 can be extended with the CIV assay module.
SUIT-008_O2_ce-pce_D025 D025 ce1 1Dig 1PM 2D 2c 3G 4S 5U 6Rot 7Ama 8AsTm 9Azd 12 ce_R ROX N_L N_P N_P N_P NS_P NS_E S_E ROX CIV ROX 8 5U 10 7Ama PM+G+S_OXPHOS+Rot_ET A: Additivity between the N- and S-pathway in the Q-junction The SUIT-008 O2 ce-pce D025 protocol is designed to assess the additivity between the N- and S-pathway in the Q-junction, providing a physiologically relevant estimate of the maximum mitochondrial respiratory capacity. It also serves as a diagnostic tool for the activity of the glutamate dehydrogenase and its linked pathways, which could be relevant in some pathologies. SUIT-008 O2 ce-pce D025 can be easily extended with the CIV assay module. In this protocol, non-permeabilized cells are added in the chamber, and ROUTINE respiration is measured. The cells are further permeabilized with digitonin inside the O2k chamber.
SUIT-008_O2_mt_D026 D026 1mt 1PM 2D 2c 3G 4S 5U 6Rot 7Ama 8AsTm 9Azd 11 ROX N_L N_P N_P N_P NS_P NS_E S_E ROX CIV ROX 7 5U 9 7Ama PM+G+S_OXPHOS+Rot_ET_mt A: Additivity between the N- and S-pathway in the Q-junction The SUIT-008 O2 mt D026 protocol can be used with mitochondrial preparations such as isolated mitochondria, tissue homogenates and permeabilized cells (already permeabilized when they are added to the chamber) in a wide variety of organisms and tissues. The protocol is designed to assess the additivity between the N- and S-pathway in the Q-junction, providing a physiologically relevant estimate of maximum mitochondrial respiratory capacity. It also serves as a diagnostic tool for the activity of the glutamate dehydrogenase and its linked pathways, which could be relevant in some pathologies. SUIT-008 O2 mt D026 can be easily extended with the CIV assay module.
SUIT-012_O2_mt_D027 D027 1mt 1PM 2D 2c 3G 4U 5Ama 7 ROX N_L N_P N_P N_P N_E ROX 6 4U 7 5Ama PM+G_OXPHOS_mt A: Coupling control (L- P- E) with NADH-linked substrates (PM and PGM) The SUIT-012 O2 mt D027 protocol specifically focuses on assessing the coupling control (L- P- E) with NADH-linked substrates (PM and PGM). Addition of G enables evaluating the contribution of this substrate in NADH-supported OXPHOS respiration, which may be relevant in the presence of glutamate-anaplerotic pathway control state. SUIT-012 can be extended with the CIV assay module.
SUIT-003_O2_ce_D028 D028 ce1 ce2Omy ce3U ce4Rot ce5S ce6Ama 6 ce_R ce_L ce_E ROX ceS_E ROX 3 ce3U 6 ce6Ama CCP-ce_+S A: Coupling control protocol, living cells »MiPNet08.09 CellRespiration Bioblast pdf SUIT-003 O2 ce D028 is a coupling-control protocol for living cells. Different coupling control states are assessed (ROUTINE - L(Omy) - E) at ce-substrate state. The use of oligomycin provides important information about the ETS and TCA activity but the optimal concentration has to be carefully determined experimentally to avoid the well-known side effects over the ET-capacity. After the addition of Rotenone to reach ROX state we add succinate, which does not permeate the plasma membrane, to test if the plasma membrane of the cells is altered (permeability test). If we do not obtain further stimulation of the respiration we could conclude that the living cells are not permeable and remain intact.
SUIT-006_O2_ce-pce_D029 D029 ce1 1Dig 1PM 2D 2c 3Omy 4U 5Ama 8 ce_R ROX N_L N_P N_P N_L N_E ROX 7 4U 8 5Ama CCP-mtprep_pce A: Coupling control protocol, mtprep SUIT-006 O2 ce-pce D029 is a coupling-control protocol for permeabilized cells. Living cells are added in the chamber, allowing to analyse ROUTINE respiration. The cells are further permeabilized inside the O2k chamber, and different coupling control states are assessed (L(n) - P - L(Omy) - E) at N(PM) pathway control state. The use of oligomycin is optional, however, it provides important information when residual and endogenous adenylates are present (which may happen if ATPases are active in the sample). This situation may lead to overestimated LEAK respiration measured in the absence of adenylates - L(n). Therefore, oligomycin can be used to verify whether this occurs and obtain the LEAK state appropriately.
SUIT-007_O2_ce-pce_D030 D030 ce1 1Dig 1G 2D 2c 3M 4U 5Ama 8 ce_R ROX N_L N_P N_P N_P N_E ROX 7 4U 8 5Ama Glutamate anaplerosis A: Glutamate anaplerotic pathway The SUIT-007 O2 pce D030 is focused on the glutamate anaplerotic pathway. If glutamate dehydrogenase is present in the sample when glutamate is added alone, it will be converted to α-ketoglutarate in an anaplerotic reaction, supporting respiration. After malate is added, it is possible to analyse the NADH Electron transfer-pathway state with glutamate and malate as substrates in the OXPHOS and Electron transfer pathway coupling control states.

In this protocol, non-permeabilized cells are added in the chamber, and ROUTINE respiration is measured. The cells are further permeabilized with digitonin inside the O2k chamber.

SUIT-020_O2_mt_D032 D032 1mt 1PM 2D 2c 3G 4S 5Rot 6Omy 7U 8Ama 10 ROX N_L N_P N_P N_P NS_P S_P S_L S_E ROX 9 7U 10 8Ama PM+G+S+Rot_OXPHOS+Omy A: simultaneous determination of O2 flux and mt-membrane potential SUIT-020 O2 mt D032 is design to assess the additivity between N-pathway and S- pathway in the Q-junction as well as investigate the N- and NS-pathway control state in mitochondrial preparations. It can serve as a diagnostic tool for the activity of glutamate dehydrogenase and its linked pathway. Oligomycin (Omy) is used to induce a LEAK state of respiration via the inhibition of the ATP synthase. Oligomycin has to be titrated in small amounts to avoid overtitration and inhibition of the flux in the ET-state. The Complex III inhibitor Antimycin A (Ama) blocks the respiration.

Originially this is a protocol for simultaneous determination of O2 flux and mitochondrial membrane potential on isolated mitochondria and tissue homogenate. It is can be used as a control experiment for SUIT-020 Fluo mt D033 without using the fluorescence dye to evaluate its effect on the O2 flux as a control.

SUIT-021_O2_mt_D035 D035 1mt 1GM 2D 2c 3S 4Rot 5Omy 6U 7Ama 9 ROX N_L N_P N_P NS_P S_P S_L S_E ROX 8 6U 9 7Ama OXPHOS (GM+S+Rot+Omy) A: simultaneous determination of O2 flux and mt-membrane potential SUIT-021 O2 mt D035 is design to assess the additivity between N-pathway and S- pathway in the Q-junction and to investigate the N- and NS-pathway control state in mitochondrial preparations . Oligomycin (Omy) is used to induce a LEAK state of respiration through inhibition of the ATP synthase. Higher concentration of Omy can decreases the ET state initiated by an uncoupler, therefore the required concentration of Omy has to be determined. Uncoupler increases the respiration and induces the ET state. Antimycin A (Ama), which is an inhibitor of Complex III), blocks the respiration.

Originially this is a protocol for simultaneous determination of O2 flux and mitochondrial membrane potential on isolated mitochondria and tissue homogenate. It can be used as a control experiment for SUIT-021 Fluo mt D036 without using the fluorescence dye in order to evaluate its effect on the O2 flux as a control.

SUIT-003_O2_ce_D037 D037 ce1 ce1Glc (ce2Omy) ce3U ce4Ama 5 ce_R ce_R ce_E ROX 4 ce3U 5 ce4Ama CCP-ce_+Glc+(Omy) A: Coupling control protocol, living cells »MiPNet08.09 CellRespiration Bioblast pdf The SUIT-003 O2 ce D037 (CCP-ce Crabtree(R)) is designed to study coupling control in living cells and the effect of glucose (or fructose; Crabtree effect) on ROUTINE respiration. This protocol allows us to omit oligomycin, therefore the LEAK-respiration. If the aim is to analyze only the Crabtree effect, it is recommended to skip the oligomycin step. If there is an interest in studying coupling control, oligomycin should be added to promote LEAK-respiration. To obtain more information about the Crabtree effect, it is ideal to use in parallel the protocol SUIT-003 O2 ce D038 (glucose addition in ET state). SUIT-003 O2 ce D037 can be extended by the cell viability test and the Complex IV module.
SUIT-003_O2_ce_D038 D038 ce1 (ce2Omy) ce3U ce3Glc ce3'U ce4Ama 6 ce_R ce_E ce_E ce_E ROX 3 ce3U 6 ce4Ama CCP-ce_+(Omy)+Glc A: Coupling control protocol, living cells »MiPNet08.09 CellRespiration Bioblast pdf SUIT-003 O2 ce D038 (CCP-ce Crabtree(E)) is designed to study coupling control in living cells and the effect of glucose (or fructose; Crabtree effect) on ET capacity. This protocol allows us to omit oligomycin, therefore the LEAK-respiration. If the aim is to analyze only the Crabtree effect, it is recommended to skip the oligomycin step. If there is an interest in studying coupling control, oligomycin should be added to promote LEAK-respiration. To obtain more information about the Crabtree effect, it is ideal to use in parallel the protocol SUIT-003 O2 ce D037 (glucose addition in ROUTINE). SUIT-003 O2 ce D038 can be extended by the cell viability test and the Complex IV module.
SUIT-003_O2_ce_D039 D039 ce1 (ce2Omy) ce3U ce4Rot ce4Ama 5 ce_R ce_E ROX ROX 3 ce3U 4 ce4Rot CCP-ce_+(Omy) A: Coupling control protocol, living cells »MiPNet08.09 CellRespiration Bioblast pdf SUIT-003 O2 ce D039 has been designed to study the coupling control state of intact microalgal cells. The different capacities are tested in the sequence OXPHOS, LEAK respiration and Electron transfer pathway. Depending on the species of microalgae studied, the oligomycin or any other inhibitor of the ATP synthase or of the adenine nucleotide translocase might not have an effect and the LEAK respiration cannot be achieved. Therefore, it is recommend to test the effect of these inhibitors in preliminary experiments for different species. Also, the inhibitory effect on the electron transfer system has to be studied to avoid an underestimation of the ET capacity. This protocol has been optimised specifically for the microalgae species Chlamydomonas reinhardtii in which oligomycin does not have an effect.
SUIT-014_O2_pfi_D042 D042 1pfi 1GM 2D 2c 3P 4S 5U 6Rot 7Ama 9 ROX N_L N_P N_P N_P NS_P NS_E S_E ROX 7 5U 9 7Ama GM+P+S_OXPHOS+Rot_ET_pfi A: Cells or tissue types that display a preference for GM over PM to support NADH-linked respiration The SUIT-014 O2 pfi D042 protocol is designed for tissue fibers that display a preference for glutamate & malate (GM) over pyruvate & malate (PM) to support NADH-linked respiration. Adding pyruvate in the OXPHOS state enables to check if NADH-linked respiration can be further stimulated to determine N-pathway P capacity. SUIT-014 protocols provide an assessment of the coupling control L- P with NADH linked-substrates (GM) and P- E with N- and S-pathways combined (PGMS). The pathway control in OXPHOS state (N, NS) and ET state (NS, S) is also evaluated, covering the contribution of two pathways which are most important in the mitochondria of many species, tissues and cell types.
SUIT-015_O2_pti_D043 D043 1pti 1OctM 2D 3G 4P 5S 6U 7Rot 8Ama 9 ROX F(N)_L F(N)_P FN_P FN_P FNS_P FNS_E S_E ROX 7 6U 9 8Ama F+G+P+S_OXPHOS+Rot_ET A: F-pathway in LEAK state and OXPHOS state SUIT-015 O2 pti D043 gives information on F-pathway in LEAK state and OXPHOS state avoiding FAO overestimation in the presence of anaplerotic pathways. In addition, the pathway control of FN and FNS in OXPHOS state and of FNS and S in ET state can be evaluated. SUIT-015 O2 pti D043 can be extended with the CIV assay module.
SUIT-016_O2_pfi_D044 D044 1pfi 1OctM 2D 3G 4S 5Rot 6Omy 7U 7c 8Ama 10 ROX F(N)_L F(N)_P FN_P FNS_P S_P S_L S_E S_E ROX 8 7U 10 8Ama F+G+S+Rot_OXPHOS+Omy A: F-pathway in LEAK state and OXPHOS state SUIT-016 O2 pfi D044 gives information on F-pathway in LEAK state and OXPHOS state in permeabized fibers, avoiding FAO overestimation in the presence of anaplerotic pathways. In addition, the pathway control of FN and FNS in OXPHOS state and of S in LEAK state and ET state is evaluated.
SUIT-019_O2_pfi_D045 D045 1pfi 1PalM 2D 2c 3Oct 4P 5G 6U 7S 8Rot 9Ama 11 ROX F(N)_L F(N)_P F(N)_P F(N)_P FN_P FN_P FN_E FNS_E S_E ROX 9 7S 11 9Ama Pal+Oct+P+G_OXPHOS+S+Rot_ET A: SUIT-019 O2 pfi D045 gives information on F-pathway in LEAK and OXPHOS states in permeabilized fibers. Moreover, this SUIT protocol allows the pathway control evaluation of FN in OXPHOS state and FN, FNS and S in ET state.
SUIT-017_O2_mt _D046 D046 1mt 1OctM 2D 2c 3G 4S 5U 6Rot 7Ama 9 ROX F(N)_L F(N)_P F(N)_P FN_P FNS_P FNS_E S_E ROX 7 5U 9 7Ama F+G+S_OXPHOS+Rot_ET_mt A: SUIT-017 O2 mt D046 gives information on F-pathway in LEAK state and OXPHOS state. In addition, the pathway control of FN and FNS in OXPHOS state and of FNS and S in ET state is evaluated.
SUIT-006_O2_mt_D047 D047 1mt 1PM 2D 2c 3Omy 4U 5Ama 7 ROX N_L N_P N_P N_L N_E ROX 6 4U 7 5Ama CCP-mtprep_PM A: Coupling control protocol, mtprep SUIT-006 O2 mt D047 is designed to study different coupling control states (L(n) - P - L(Omy) - E) at N(PM) pathway control state. The use of oligomycin is optional, however, it provides important information when endogenous adenylates are present and ATPases are active in the sample. This situation may lead to overestimated LEAK respiration measured in the absence of adenylates - L(n). Therefore, oligomycin can be used to verify whether this occurs and obtain the LEAK state appropriately.
SUIT-017_O2_pfi_D049 D049 1pfi 1OctM 2D 2c 3G 4S 5U 6Rot 7Ama 9 ROX F(N)_L F(N)_P F(N)_P FN_P FNS_P FNS_E S_E ROX 7 5U 9 7Ama F+G+S_OXPHOS+Rot_ET_pfi A: SUIT-017 O2 pfi D049 gives information on F-pathway in LEAK state and OXPHOS state avoiding fatty acid oxidation (FAO) overestimation in the presence of anaplerotic pathways. In addition, the pathway control of FN and FNS in OXPHOS state and of FNS and S in ET state is evaluated.
SUIT-003_O2_ce_D050 D050 ce1 ce1Snv ce2Omy ce3U ce4Rot ce5Ama 6 ce_R ce_R ce_E ROX ROX 4 ce3U 6 ce5Ama CCP-ce_+Snv+(Omy) A: Coupling control protocol, living cells »MiPNet08.09 CellRespiration Bioblast pdf SUIT-003 O2 ce D050 protocol is designed to study CI-deficiency in living cells. Plasma membrane-permeable succinate (MitoKit-CII/Succinate-nv, Snv), which is added in ROUTINE stimulates mitochondrial respiration in CI-deficient human blood cells, fibroblasts and heart fibres, acting on Complex II of the electron transfer system. Moreover, the SUIT-003 O2 ce D050 protocol enables evaluation of the coupling control of living cells. Respiratory capacities are tested in a sequence of coupling states: ROUTINE and Electron transfer pathway. Optionally, to study LEAK respiration, the phosphorylation system is inhibited by oligomycin. The final concentration of oligomycin has to be carefully optimized for various cell types, to minimize the inhibitory effect on the electron transfer system which would lead to an underestimation of ET capacity.
SUIT-022 O2 ce D051 D051 ce1 ce2KCN ce3SHAM 3 ce_R ce_R ROX 3 ce3SHAM 3 ce3SHAM AOX (ce CN+SHAM) A: Determination of the respiration due to the alternative oxidase pathway The SUIT-022 O2 ce D051 provides an estimation of mitochondrial respiration due to the Q-alternative oxidase pathway (Q-AOX pathway), which is cyanide-and antimycin A-resistant, for the specific case of microalgal cells. This protocol must be complemented with the study in parallel of mitochondrial respiration through the CIII- cytochrome c oxidase pathway (CIII-CIV; see SUIT-023).
SUIT-012_O2_ce-pce_D052 D052 ce1 1Dig 1PM 2D 2c 3G 4U 5Ama 8 ce_R ROX N_L N_P N_P N_P N_E ROX 8 5Ama 7 4U PM+G_OXPHOS_pce A: Coupling control (L- P- E) with NADH-linked substrates (PM and PGM) The SUIT-012 O2 ce-pce D052 protocol specifically focuses on assessing the coupling control (L- P- E) with NADH-linked substrates (PM and PGM). Addition of G enables evaluating the contribution of this substrate in NADH-supported OXPHOS respiration, which may be relevant in the presence of glutamate-anaplerotic pathway control state. SUIT-012 can be extended with the CIV assay module.
SUIT-023 O2 ce D053 D053 ce1 ce2SHAM ce3KCN 3 ce_R ce_R ROX 3 ce3KCN 3 ce3KCN AOX-ce SHAM+CN A: Determination of respiration through the CIII-CIV pathway The SUIT-023 O2 ce D053 provides an estimation of mitochondrial respiration in microalgal living cells through the CIII-cytochrome c oxidase pathway (CIII-CIV), which is inhibited by antimycin A and cyanide. This protocol must be complemented with the study in parallel of the mitochondrial respiration through the Q-alternative oxidase pathway (see SUIT-022).
SUIT-018_O2_mt_D054 D054 1mt 1GMS 2D 3Ama 4 ROX NS_L NS_P ROX 3 2D 4 3Ama O2_GMS_mt4 SUIT-018 O2 mt D054 is a short protocol to study the influence of a fluorescence dye on the O2 flux. During this protocol the [O2] can be modified by injecting nitrogen in the chamber.
SUIT-006_MgG_mt_D055 D055 1mt 1PM 2D 3Cat 4U 5Ama 6 ROX N_L N_P N_L N_E ROX 5 4U 6 5Ama SUIT-006 MgG mt D055 is a protocol to investigate the O2 flux and mitochondrial ATP production using the fluorescent dye Magnesium Green. In this protocol, the NADH Electron transfer-pathway state can be analyzed in mitochondrial preparations such as isolated mitochondria, tissue homogenates and permeabilized cells (already permeabilized when they are added to the chamber).

Addition of the substrates pyruvate & malate (PM) to the mitochondrial preparation leads to LEAK state, and further addition of ADP induces the OXPHOS state, in which respiration is coupled to ATP production. After ADP addition, the signal from MgG gradually decreases, reflecting the ANT-mediated exchange of ADP for ATP, the later one having higher affinity for Mg2+. This exchange is stopped by addition of the ANT inhibitor carboxyatractyloside (Cat), which leads to LEAK state. With the addition of uncoupler, the Electron transfer pathway state can also be assessed in this protocol.

In the DatLab software, SUIT-006 MgG mt D055 DLP file is currently provided for the category N(PM). For using this protocol with other substrate/inhibitor combinations (e.g. succinate and rotenone for Succinate pathway control state or PMS for NS-pathway control state), a personalized DLPU can be created.

SUIT-024_O2_ce-pce_D056 D056 ce1 1Dig 1PM 2T 2D 3Omy 4Ama 7 ce_R ROX N_L N_P N_P N_L ROX 5 2D 7 4Ama ATPase (PM) A: Determination of the presence of ATPases in mitochondrial preparations. SUIT-006 MgG mt D055 is a protocol to investigate the O2 flux and mitochondrial ATP production using the fluorescent dye Magnesium Green. In this protocol, the NADH Electron transfer-pathway state can be analyzed in mitochondrial preparations such as isolated mitochondria, tissue homogenates and permeabilized cells (already permeabilized when they are added to the chamber).

Addition of the substrates pyruvate & malate (PM) to the mitochondrial preparation leads to LEAK state, and further addition of ADP induces the OXPHOS state, in which respiration is coupled to ATP production. After ADP addition, the signal from MgG gradually decreases, reflecting the ANT-mediated exchange of ADP for ATP, the later one having higher affinity for Mg2+. This exchange is stopped by addition of the ANT inhibitor carboxyatractyloside (Cat), which leads to LEAK state. With the addition of uncoupler, the Electron transfer pathway state can also be assessed in this protocol.

In the DatLab software, SUIT-006 MgG mt D055 DLP file is currently provided for the category N(PM). For using this protocol with other substrate/inhibitor combinations (e.g. succinate and rotenone for Succinate pathway control state or PMS for NS-pathway control state), a personalized DLPU can be created.

SUIT-025_O2_mt_D057 D057 1mt 1D 2M.1 3Oct 3c 4M2 5P 6G 7S 8Rot 9Ama 11 ROX ROX non F(N)_P F(N)_P F(N)_P F(N)_P FN_P FN_P FNS_P S_P ROX 9 7S 11 9Ama OXPHOS (F+M+P+G+S+Rot) The SUIT-025 O2 mt D057 protocol is based on SUIT-002 specially designed to give information on F-pathway in OXPHOS state avoiding fatty acid oxidation (FAO) overestimation in the presence of anaplerotic pathways. Moreover, the pathway control in OXPHOS state (F, F(N), FN, FNS, pathways) can be evaluated by using this SUIT protocol.
SUIT-003_O2_ce_D060 D060 ce1 (ce2Omy) ce3U ce4Rot ce5Snv ce6Mnanv ce7Ama 7 ce_R ceP_E ROX ceS_E ROX ROX 3 ce3U 7 ce7Ama CCP-ce_+(Omy)+Snv+Mnanv A: Coupling control protocol, living cells »MiPNet08.09 CellRespiration Bioblast pdf In SUIT-003 O2 ce D060 protocol, the use of permeable succinate and malonate enables further information to be obtained about the mitochondrial respiratory capacities in living cells. The plasma membrane-permeable compounds NV118/MitoKit-CII (Snv) and NV161/MitoKit-CII (Mnanv) require cleavage by esterases present in the cytosol to release respectively succinate and malonate, which will happen therefore only in living cells with intact plasma membrane. The use of Snv after rotenone provides information about the S-pathway in physiological conditions. Rotenone inhibits Complex I and consequently the TCA cycle and respiration in living cells, but intracellular release of succinate from the permeable compound may lead to stimulation of respiration. Afterwards, the addition of Mnanv will counteract this effect. Moreover, the SUIT-003 O2 ce D060 protocol enables evaluation of the coupling control of living cells. Respiratory capacities are tested in a sequence of coupling states: ROUTINE and Electron transfer pathway. Optionally, to study LEAK respiration, the phosphorylation system might be inhibited by oligomycin. The final concentration of oligomycin has to be carefully optimized for various cell types, to minimize the inhibitory effect on the electron transfer system which would lead to an underestimation of ET capacity.
SUIT-003_O2_ce_D061 D061 ce1 (ce2Omy) ce3U ce4Rot ce5DMSO ce6DMSO ce7Ama 7 ce_R ceP_E ROX ROX ROX ROX 3 ce3U 7 ce7Ama CCP-ce_+(Omy)+DMSO+DMSO A: Coupling control protocol, living cells »MiPNet08.09 CellRespiration Bioblast pdf SUIT-003 O2 ce D061 protocol is a control for the SUIT-003 O2 ce D060, with the addition of the carrier DMSO instead of the plasma membrane-permeable succinate (MitoKit-CII/Succinate-nv) and plasma membrane-permeable malonate (MitoKit-CII/Malonate-nv). Moreover, the SUIT-003 O2 ce D061 protocol allows evaluating the coupling control of living cells. Respiratory capacities are tested in a sequence of coupling states: ROUTINE and Electron transfer pathway. Optionally, to study LEAK respiration, the phosphorylation system might be inhibited by oligomycin. The final concentration of oligomycin has to be carefully optimized for various cell types, to minimize the inhibitory effect on the electron transfer system which would lead to an underestimation of ET capacity.
SUIT-003_O2_ce_D062 D062 ce1 ce1DMSO (ce2Omy) ce3U ce4Rot ce5Ama 6 ce_R ce_R ce_E ROX ROX 4 ce3U 6 ce5Ama CCP-ce_+DMSO+(Omy) A: Coupling control protocol, living cells »MiPNet08.09 CellRespiration Bioblast pdf SUIT-003 O2 ce D062 protocol is the control of the SUIT-003 O2 ce D050 adding the carrier DMSO in ROUTINE respiration instead of the plasma membrane-permeable succinate (MitoKit-CII/Succinate-nv). Moreover,the SUIT-003 O2 ce D062 protocol allows to evaluate the coupling control of living cells. Respiratory capacities are tested in a sequence of coupling states: ROUTINE and Electron transfer pathway. Optionally, to study LEAK respiration, the phosphorylation system might be inhibited by oligomycin. The final concentration of oligomycin has to be carefully optimized for various cell types, to minimize the inhibitory effect on the electron transfer system which would lead to an underestimation of ET capacity.
SUIT-026_O2_mt_D063 D063 1mt 1S 2Rot 3D 3c 4Ama 6 ROX S_L S_L S_P S_P ROX 4 3D 6 4Ama S+Rot_OXPHOS SUIT-026 O2 mt D063 has been designed to serve as a respiratory control of the SUIT-026 AmR mt D064 (protocol to study the RET-initiated ROS production in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells which are already permeabilized when they are added to the chamber).
SUIT-027_O2_ce-pce_D065 D065 ce1 1Dig 1M.05 2D 3M 3M´ 4P 5G 8 ce_R ROX (N)_P (N)_P N_P N_P 8 5G 2 1Dig Malate anaplerosis A: Malate anaplerotic pathway The SUIT-027 O2 ce-pce D065 is focused on the analysis of the malate-anaplerotic pathway control state in mitochondrial preparations (isolated mitochondria, permeabilized cells, homogenate, and permeabilized fibers). Malate alone does not support respiration if oxaloacetate is not metabolized further in the absence of acetyl-CoA. The careful titration of malate is required to analyze the activity of the mt-isoform of NADP+- or NAD(P)+-dependent malic enzyme (mtME). If these enzymes are present in the sample one should be able to reach with malate alone a high respiratory activity comparable to the NADH-linked pathway control states with more classical combinations of substrates (e.g. PM or GM).

Protocol Marks

Protocol name DLP# Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Step 7 Step 8 Step 9 Step 10 Step 11 Step 12 Step 13 Step 14 Step 15 Step 16 Step 17 Step 18 Step 19 Step 20 # of steps
SUIT-001_O2_ce-pce_D003 D003 ce1 1Dig 1PM 2D 2c 3U 4G 5S 6Oct 7Rot 8Gp 9Ama 10AsTm 11Azd 14
SUIT-001_O2_PBMC-PLT_D004 D004 ce1 1Dig 1PM 2D 2c 3U 4G 5S (6Oct) 7Rot 8Gp 9Ama 10AsTm 11Azd 14
SUIT-001_O2_mt_D001 D001 1mt 1PM 2D 2c 3U 4G 5S 6Oct 7Rot 8Gp 9Ama 10AsTm 11Azd 13
SUIT-001_O2_pfi_D002 D002 1pfi 1PM 2D 2c 3U 4G 5S 6Oct 7Rot 8Gp 9Ama 10AsTm 11Azd 13
SUIT-002_O2_ce-pce_D007 D007 ce1 1Dig 1D 2M.1 3Oct 3c 4M2 5P 6G 7S 8Gp 9U 10Rot 11Ama 12AsTm 13Azd 16
SUIT-002_O2_ce-pce-PBMC-PLT_D007a D007a ce1 1Dig 1D 2M.1 3Oct 3c 4M2 5P 6G 7S 8Gp 9U 10Rot 11Ama 12AsTm 13Azd 16
SUIT-002_O2_mt_D005 D005 1mt 1D 2M.1 3Oct 3c 4M2 5P 6G 7S 8Gp 9U 10Rot 11Ama 12AsTm 13Azd 15
SUIT-002_O2_pfi_D006 D006 1pfi 1D 2M.1 3Oct 3c 4M2 5P 6G 7S 8Gp 9U 10Rot 11Ama 12AsTm 13Azd 15
SUIT-003_O2_ce_D009 D009 ce1 ce2Omy ce3U ce4Ama 4
SUIT-003_O2_ce_D012 D012 ce1 ce2P ce2Omy ce3U ce4Rot ce5Ama 6
SUIT-003_O2_ce_D028 D028 ce1 ce2Omy ce3U ce4Rot ce5S ce6Ama 6
SUIT-003_O2_ce_D037 D037 ce1 ce1Glc ce2Omy ce3U ce4Ama 5
SUIT-003_O2_ce_D038 D038 ce1 ce2Omy ce3U ce3Glc ce3'U ce4Ama 6
SUIT-003_O2_ce_D039 D039 ce1 ce2Omy ce3U ce4Rot ce4Ama 5
SUIT-003_O2_ce_D050 D050 ce1 ce1Snv ce2Omy ce3U ce4Rot ce5Ama 6
SUIT-003_O2_ce_D060 D060 ce1 ce2Omy ce3U ce4Rot ce5Snv ce6Mnanv ce7Ama 7
SUIT-003_O2_ce_D061 D061 ce1 ce2Omy ce3U ce4Rot ce5DMSO ce6DMSO ce7Ama 7
SUIT-003_O2_ce_D062 D062 ce1 ce1DMSO ce2Omy ce3U ce4Rot ce5Ama 6
SUIT-003_O2_ce-pce_D013 D013 ce1 ce1P ce2Omy ce3U ce3Glc ce3M ce4Rot ce5S 1Dig 1U 1c 2Ama 3AsTm 4Azd 14
SUIT-003_O2_ce-pce_D018 D018 ce1 ce1P ce2Omy ce3U ce3Glc ce4Rot ce5S 1Dig 1c 2Ama 3AsTm 4Azd 12
SUIT-003_O2_ce-pce_D020 D020 ce1 ce1P ce2Omy ce3U ce4Rot ce5S 1Dig 1c 2Ama 3AsTm 4Azd 11
SUIT-004_O2_pfi_D010 D010 1pfi 1PM 2D 2c 3U 4S 5Rot 6Ama 7AsTm 8Azd 10
SUIT-005_O2_pfi_D011 D011 1pfi 1OctM 2D 2c 3P 4S 5U 6Rot 7Ama 8AsTm 9Azd 11
SUIT-006_O2_ce-pce_D029 D029 ce1 1Dig 1PM 2D 2c 3Omy 4U 5Ama 8
SUIT-006_O2_mt_D022 D022 1mt 1S(Rot) 2D 2c 3Omy 4U 5Ama 7
SUIT-006_O2_mt_D047 D047 1mt 1PM 2D 2c 3Omy 4U 5Ama 7
SUIT-007_O2_ce-pce_D030 D030 ce1 1Dig 1G 2D 2c 3M 4U 5Ama 8
SUIT-008_O2_ce-pce_D025 D025 ce1 1Dig 1PM 2D 2c 3G 4S 5U 6Rot 7Ama 8AsTm 9Azd 12
SUIT-008_O2_mt_D026 D026 1mt 1PM 2D 2c 3G 4S 5U 6Rot 7Ama 8AsTm 9Azd 11
SUIT-008_O2_pfi_D014 D014 1pfi 1PM 2D 2c 3G 4S 5U 6Rot 7Ama 8AsTm 9Azd 11
SUIT-009_O2_ce-pce_D016 D016 ce1 1Dig 1S 2D 2c 3P 4Rot 5Ama 8
SUIT-009_O2_mt_D015 D015 1mt 1S 2D 2c 3P 4Rot 5Ama 7
SUIT-010_O2_ce-pce_D008 D008 ce1 ce2Rot ce3S ce4D 1Dig 1c 6
SUIT-011_O2_pfi_D024 D024 1pfi 1GM 2D 2c 3S 4U 5Rot 6Ama 8
SUIT-012_O2_ce-pce_D052 D052 ce1 1Dig 1PM 2D 2c 3G 4U 5Ama 8
SUIT-012_O2_mt_D027 D027 1mt 1PM 2D 2c 3G 4U 5Ama 7
SUIT-014_O2_pfi_D042 D042 1pfi 1GM 2D 2c 3P 4S 5U 6Rot 7Ama 9
SUIT-015_O2_pti_D043 D043 1pti 1OctM 2D 3G 4P 5S 6U 7Rot 8Ama 9
SUIT-016_O2_pfi_D044 D044 1pfi 1OctM 2D 3G 4S 5Rot 6Omy 7U 7c 8Ama 10
SUIT-017_O2_mt _D046 D046 1mt 1OctM 2D 2c 3G 4S 5U 6Rot 7Ama 9
SUIT-017_O2_pfi_D049 D049 1pfi 1OctM 2D 2c 3G 4S 5U 6Rot 7Ama 9
SUIT-018_O2_mt_D054 D054 1mt 1GMS 2D 3Ama 4
SUIT-019_O2_pfi_D045 D045 1pfi 1PalM 2D 2c 3Oct 4P 5G 6U 7S 8Rot 9Ama 11
SUIT-020_O2_mt_D032 D032 ROX N_L N_P N_P N_P NS_P S_P S_L S_E ROX 10
SUIT-021_O2_mt_D035 D035 1mt 1GM 2D 2c 3S 4Rot 5Omy 6U 7Ama 9
SUIT-022 O2 ce D051 D051 ce1 ce2KCN ce3SHAM 3
SUIT-023 O2 ce D053 D053 ce1 ce2SHAM ce3KCN 3
SUIT-024_O2_ce-pce_D056 D056 ce1 1Dig 1PM 2T 2D 3Omy 4Ama 7
SUIT-025_O2_mt_D057 D057 1mt 1D 2M.1 03-Oct 3c 4M2 5P 6G 7S 8Rot 9Ama 11
SUIT-026_O2_mt_D063 D063 1mt 1S 2Rot 3D 3c 4Ama 6
SUIT-027_O2_ce-pce_D065 D065 ce1 1Dig 1M.05 2D 3M 3M´ 4P 5G 8

Respiratory states

DLP# State 1 State 2 State 3 State 4 State 5 State 6 State 7 State 8 State 9 State 10 State 11 State 12 State 13 State 14 State 15 State 16 State 17 State 18 State 19 State 20
D003 ce_R ROX N_L N_P N_P N_E N_E NS_E FNS_E S_E SGp_E ROX CIV ROX
D004 ce_R ROX N_L N_P N_P N_E N_E NS_E S_E SGp_E ROX CIV ROX
D001 ROX N_L N_P N_P N_E N_E NS_E FNS_E S_E SGp_E ROX CIV ROX
D002 ROX N_L N_P N_P N_E N_E NS_E FNS_E S_E SGp_E ROX CIV ROX
D007 ce_R ROX ROX F_P F_P F(N)_P FN_P FN_P FNS_P FNSGp_P FNSGp_E SGp_E ROX CIV ROX
D007a ce_R ROX ROX F_P F_P F(N)_P FN_P FN_P FNS_P FNSGp_P FNSGp_E SGp_E ROX CIV ROX
D005 ROX ROX F_P F_P F(N)_P FN_P FN_P FNS_P FNSGp_P FNSGp_E SGp_E ROX CIV ROX
D006 ROX ROX F_P F_P F(N)_P FN_P FN_P FNS_P FNSGp_P FNSGp_E SGp_E ROX CIV ROX
D009 ce_R ce_L ce_E ROX
D012 ce_R ce_R ce_L ce_E ROX ROX
D028 ce_R ce_L ce_E ROX ceS_E ROX
D037 ce_R ce_R ce_E ROX
D038 ce_R ce_E ce_E ce_E ROX
D039 ce_R ET ROX ROX
D050 ce_R ce_R ce_E ROX ROX
D060 ce_R ceP_E ROX ceS_E ROX ROX
D061 ce_R ceP_E ROX ROX ROX ROX
D062 ce_R ce_R ce_E ROX ROX
D013 ce_R ce_R ce_L ce_E ce_E ce_E ROX ceS_E S_E S_E S_E ROX CIV ROX
D018 ce_R ce_R ce_L ce_E ce_E ROX ceS_E S_E S_E ROX CIV ROX
D020 ce_R ce_R ce_L ce_E ROX ceS_E S_E S_E ROX CIV ROX
D010 ROX N_L N_P N_P N_E NS_E S_E ROX CIV ROX
D011 ROX F(N)_L F(N)_P F(N)_P FN_P FNS_P FNS_E S_E ROX CIV ROX
D029 ce_R ROX N_L N_P N_P N_L N_E ROX
D022 ROX S_L S_P S_P S_E ROX
D047 ROX N_L N_P N_P N_L N_E ROX
D030 ce_R ROX N_L N_P N_P N_P N_E ROX
D025 ce_R ROX N_L N_P N_P N_P NS_P NS_E S_E ROX CIV ROX
D026 ROX N_L N_P N_P N_P NS_P NS_E S_E ROX CIV ROX
D014 ROX N_L N_P N_P N_P NS_P NS_E S_E ROX CIV ROX
D016 ce_R ROX S_L S_P S_P NS_P S_P ROX
D015 ROX S_L S_P S_P NS_P S_P ROX
D008 ce_R ROX ceS_L ceS_P S_P S_P
D024 ROX N_L N_P N_P NS_P NS_E S_E ROX
D052 ce_R ROX N_L N_P N_P N_P N_E ROX
D027 ROX N_L N_P N_P N_P N_E ROX
D042 ROX N_L N_P N_P N_P NS_P NS_E S_E ROX
D043 ROX F(N)_L F(N)_P FN_P FN_P FNS_P FNS_E S_E ROX
D044 ROX F(N)_L F(N)_P FN_P FNS_P S_P S_L S_E S_E ROX
D046 ROX F(N)_L F(N)_P F(N)_P FN_P FNS_P FNS_E S_E ROX
D049 ROX F(N)_L F(N)_P F(N)_P FN_P FNS_P FNS_E S_E ROX
D054 ROX NS_L NS_P ROX
D045 F(N)_L F(N)_P F(N)_P F(N)_P FN_P FN_P FN_E FNS_E S_E ROX
D032 ROX N_L N_P N_P N_P NS_P S_P S_L S_E ROX
D035 ROX N_L N_P N_P NS_P S_P S_L S_E ROX
D051 ce_R ce_R ROX
D053 ce_R ce_R ROX
D056 ce_R ROX N_L N_P N_P N_L ROX
D057 ROX ROX non F(N)_P F(N)_P F(N)_P F(N)_P FN_P FN_P FNS_P S_P ROX
D063 ROX S_L S_L S_P S_P ROX
D065 ce_R ROX (N)_P (N)_P N_P N_P

Short names and notes

DLP# Short name Notes
D003 RP1_pce Doerrier 2018 Methods Mol Biol
D004 RP1_PBMC-PLT Doerrier 2018 Methods Mol Biol
D001 RP1_mt Doerrier 2018 Methods Mol Biol
D002 RP1_pfi Doerrier 2018 Methods Mol Biol
D007 RP2_pce A: Doerrier 2018 Methods Mol Biol
D007a RP2_PBMC-PLT A: Doerrier 2018 Methods Mol Biol
D005 RP2_mt A: Doerrier 2018 Methods Mol Biol
D006 RP2_pfi A: Doerrier 2018 Methods Mol Biol
D009 CCP-ce A: Coupling control protocol, living cells »MiPNet08.09 CellRespiration Bioblast pdf
D012 CCP-ce_+P A: Coupling control protocol, living cells »MiPNet08.09 CellRespiration Bioblast pdf
D028 CCP-ce_+S A: Coupling control protocol, living cells »MiPNet08.09 CellRespiration Bioblast pdf
D037 CCP-ce_+Glc+(Omy) A: Coupling control protocol, living cells »MiPNet08.09 CellRespiration Bioblast pdf
D038 CCP-ce_+(Omy)+Glc A: Coupling control protocol, living cells »MiPNet08.09 CellRespiration Bioblast pdf
D039 CCP-ce_+(Omy) A: Coupling control protocol, living cells »MiPNet08.09 CellRespiration Bioblast pdf
D050 CCP-ce_+Snv+(Omy) A: Coupling control protocol, living cells »MiPNet08.09 CellRespiration Bioblast pdf
D060 CCP-ce_+(Omy)+Snv+Mnanv A: Coupling control protocol, living cells »MiPNet08.09 CellRespiration Bioblast pdf
D061 CCP-ce_+(Omy)+DMSO+DMSO A: Coupling control protocol, living cells »MiPNet08.09 CellRespiration Bioblast pdf
D062 CCP-ce_+DMSO+(Omy) A: Coupling control protocol, living cells »MiPNet08.09 CellRespiration Bioblast pdf
D013 CCP-ce_+Glc+M+viability+CIV A: Coupling control protocol, living cells »MiPNet08.09 CellRespiration Bioblast pdf
D018 CCP-ce_+Glc+viability+CIV A: Coupling control protocol, living cells »MiPNet08.09 CellRespiration Bioblast pdf
D020 CCP-ce_+viability+CIV A: Coupling control protocol, living cells »MiPNet08.09 CellRespiration Bioblast pdf
D010 RP1-short
D011 RP2-short A: when malate-anaplerotic activity is zero
D029 CCP-mtprep_pce A: Coupling control protocol, mtprep
D022 CCP-mtprep_Srot A: Coupling control protocol, mtprep
D047 CCP-mtprep_PM A: Coupling control protocol, mtprep
D030 Glutamate anaplerosis A: Glutamate anaplerotic pathway
D025 PM+G+S_OXPHOS+Rot_ET A: Additivity between the N- and S-pathway in the Q-junction
D026 PM+G+S_OXPHOS+Rot_ET_mt A: Additivity between the N- and S-pathway in the Q-junction
D014 PM+G+S_OXPHOS+Rot_ET_pfi A: Additivity between the N- and S-pathway in the Q-junction
D016 S_OXPHOS_Rot_O2_pce
D015 S_OXPHOS_Rot_O2_mt
D008 Digitonin test A: Optimization of digitonin concentration for pce
D024 GM+S_OXPHOS+Rot_ET:pfi A: Maximum mitochondrial respiratory capacity (OXPHOS with NS substrates) and coupling/pathway control
D052 PM+G_OXPHOS_pce A: Coupling control (L- P- E) with NADH-linked substrates (PM and PGM)
D027 PM+G_OXPHOS_mt A: Coupling control (L- P- E) with NADH-linked substrates (PM and PGM)
D042 GM+P+S_OXPHOS+Rot_ET_pfi A: Cells or tissue types that display a preference for GM over PM to support NADH-linked respiration
D043 F+G+P+S_OXPHOS+Rot_ET A: F-pathway in LEAK state and OXPHOS state
D044 F+G+S+Rot_OXPHOS+Omy A: F-pathway in LEAK state and OXPHOS state
D046 F+G+S_OXPHOS+Rot_ET_mt A:
D049 F+G+S_OXPHOS+Rot_ET_pfi A:
D054 O2_GMS_mt4
D045 Pal+Oct+P+G_OXPHOS+S+Rot_ET A:
D032 PM+G+S+Rot_OXPHOS+Omy A: simultaneous determination of O2 flux and mt-membrane potential
D035 OXPHOS (GM+S+Rot+Omy) A: simultaneous determination of O2 flux and mt-membrane potential
D051 AOX (ce CN+SHAM) A: Determination of the respiration due to the alternative oxidase pathway
D053 AOX-ce SHAM+CN A: Determination of respiration through the CIII-CIV pathway
D056 ATPase (PM) A: Determination of the presence of ATPases in mitochondrial preparations.
D057 OXPHOS (F+M+P+G+S+Rot)
D063 S+Rot_OXPHOS
D065 Malate anaplerosis A: Malate anaplerotic pathway

Reference States

DLP# Reference st. Mark Baseline st. Mark
D003 8 5S 12 9Ama
D004 8 5S 12 9Ama
D001 7 5S 11 9Ama
D002 7 5S 11 9Ama
D007 12 9U 14 11Ama
D007a 12 9U 14 11Ama
D005 11 9U 13 11Ama
D006 11 9U 13 11Ama
D009 3 ce3U 4 ce4Ama
D012 4 ce3U 6 ce5Ama
D028 3 ce3U 6 ce6Ama
D037 4 ce3U 5 ce4Ama
D038 3 ce3U 6 ce4Ama
D039 3 ce3U 4 ce4Rot
D050 4 ce3U 6 ce5Ama
D060 3 ce3U 7 ce7Ama
D061 3 ce3U 7 ce7Ama
D062 4 ce3U 6 ce5Ama
D013 4 ce3U 12 2Ama
D018 4 ce3U 10 2Ama
D020 4 ce3U 9 2Ama
D010 6 4S 8 6Ama
D011 7 5U 9 7Ama
D029 7 4U 8 5Ama
D022 6 4U 7 5Ama
D047 6 4U 7 5Ama
D030 7 4U 8 5Ama
D025 8 5U 10 7Ama
D026 7 5U 9 7Ama
D014 7 5U 9 7Ama
D016 6 3P 8 5Ama
D015 5 3P 7 5Ama
D008 5 1Dig 2 ce2Rot
D024 6 4U 8 6Ama
D052 8 4U 7 5Ama
D027 6 4U 7 5Ama
D042 7 5U 9 7Ama
D043 7 6U 9 8Ama
D044 8 7U 10 8Ama
D046 7 5U 9 7Ama
D049 7 5U 9 7Ama
D054 3 2D 4 3Ama
D045 9 7S 11 9Ama
D032 9 7U 10 8Ama
D035 8 6U 9 7Ama
D051 1 ce1 3 ce3SHAM
D053 1 ce1 3 ce3KCN
D056 5 2D 7 4Ama
D057 9 7S 11 9Ama
D063 4 3D 6 4Ama
D065 8 5G 2 1Dig