MitoXpress FAO Assay. For the measurement of Fatty Acid Oxidation

Transcription

1 MitoXpress FAO Assay For the measurement of Fatty Acid Oxidation Companion Kit to be used in combination with Luxcel s MitoXpress Xtra - Oxygen Consumption Assay ILLUMINATING DISCOVERY

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3 TABLE OF CONTENTS GENERAL INFORMATION MATERIALS SUPPLIED ADDITIONAL ITEMS REQUIRED OPTIONAL ITEMS NOT SUPPLIED DESCRIPTION TYPICAL WORKFLOW PERFORMING THE MITOXPRESS FAO ASSAY CELL CULTURE AND PLATING PRE-ASSAY PREPARATION GLUCOSE DEPRIVATION STEP (OPTIONAL) TYPICAL ASSAY OPTIONAL CONTROLS ANALYSIS SAMPLE FAO-DRIVEN OXYGEN CONSUMPTION EVALUATING EXOGENOUS AND ENDOGENOUS FAO METABOLIC CHARACTERISATION APPENDIX A - EXPLANATORY NOTES REFERENCES RELATED PRODUCTS... 14

4 GENERAL INFORMATION MATERIALS SUPPLIED Assay kit will arrive at room temperature. For best results store individual components as indicated below. The kit is designed for use with MitoXpress Xtra - Oxygen Consumption Assay (MX-200). Cat. # Comp. # Kit Component Desc. Contents Storage No of Tests 1 MXC-500 MXC-501 Oleate-BSA conjugate 1 vial 1 ml of Oleate (3 mm Oleate) +4 C 200 MXC-502 BSA control 1 vial 0.5 ml of BSA ( 1.5 mm) +4 C 100 MXC-503 Base Measurement 1 tablet Base media tablet Media Tablet (KHB) 2 RT 200 MXC-504 L-Carnitine 1 vial 4 mg L-Carnitine hydrochloride +4 C 200 MXC-505 FCCP 1 vial mg -20 C 055 MXC-506 Etomoxir 1 vial mg -20 C Number of tests based on described procedure, stock dilutions, concentrations and wash steps. 2 Tablet formulation: 111 mm NaCl, 4.7 mm KCL, 2 mm MgSO4, 1.25 mm CaCl2, 1.2 mm NaH2PO4. NOTE: On receipt each MitoXpress FAO component should be stored as indicated above. A labelled zip-lock bag is provided for convenient storage at -20 C. ADDITIONAL ITEMS REQUIRED MitoXpress Xtra HS - Oxygen Consumption Assay Kit (MX-200) Standard clear 96-well TC + plates OR 96-well black wall clear bottom TC + plates Fluorescence plate reader, with suitable filters and plate temperature control Components for glucose deprivation media: (Glucose free DMEM (Sigma # D5030), glucose, L-glutamine, foetal bovine serum, pen/strep). OPTIONAL ITEMS NOT SUPPLIED Plate block heater for plate preparation SUPPORT Visit our website P3

5 DESCRIPTION Fatty acid oxidation (FAO) is the primary metabolic pathway in a variety of tissues, becoming particularly important during periods of glucose deprivation. In organs such as liver and skeletal muscle, FAO can provide over 75% of cellular ATP while in cardiac tissue it can be responsible for up to 90% of cellular energy requirements [1, 2] FAO is also now acknowledged as a key factor in cancer metabolism [3] and is also implicated in drug-induced microsteatosis [4]. To facilitate the convenient measurement of FAO-driven respiration, Luxcel Biosciences has developed the MitoXpress Xtra FAO Kit (MXC-500). The kit is designed for use with the MitoXpress Xtra HS - Oxygen Consumption Assay (MX-200) and contains the 18C unsaturated fatty acid Oleate as substrate, supplied as a 2:1 BSA conjugate. The kit also contains a buffer tablet and L-Carnitine for convenient preparation of measurement media and two FAO modulators, Etomoxir and FCCP. Etomoxir inhibits CPT1 thereby preventing Oleate import (Fig. 1), limiting the supply of reducing equivalents to the ETC, and, in turn, reducing oxygen consumption. The remaining ETC activity is driven by non-long chain FAO. FCCP treatment induces maximal ETC activity by dissipating the mitochondrial membrane potential, while the increased demand for reducing equivalents causes a concomitant increases in FAO activity. This increase is limited where exogenous LCFA is unavailable or where import is inhibited. The primary pathway for the degradation of fatty acids is mitochondrial fatty acid β oxidation. Longchain fatty acids (LCFAs) are imported into the mitochondria as acyl carnitine. Once inside, acyl- CoAs are released to undergo an iterative four-step oxidation until the entire chain is oxidized to acetyl- CoA, while carnitine returns to facilitate further LCFA transport. The Acetyl CoA produced typically enters the TCA, although in liver it can also fuel the production of ketone bodies, an important energy source for other tissues. Both TCA and β oxidation contribute to the pool of reducing equivalents (NADH and FADH2) which, in turn, drive the activity of the ETC and subsequent ATP generation. Figure 1: Overview of long-chain fatty acid activation, import and oxidation P4

6 TYPICAL WORKFLOW DAY 1 Plate cells (2D or 3D) and return to culture over night Prepare Base Measurement Media from Base Management Media tablet DAY 2 (Optional) Prepare Glucose-Deprivation Media Replace culture media with Glucose-Deprivation Media and return to culture Prepare FA-Free and FA Measurement Media from Base Measurement Media Prepare MitoXpress FAO kit controls (Etoxomir, FCCP and BSA) Prepare MitoXpress Xtra reagent (MX-200) DAY 3 Aspirate spent media and wash twice with FA-Free Measurement Media Add FA or FA-free Measurement Media and MitoXpress - Xtra reagent Add controls (Etoxomir, FCCP, BSA) as pre-optimised concentrations and timings Overlay plate with HS mineral oil (MX-200) Measure on fluorescence plate reader using recommended settings Analyse kinetic data output to determine FAO-driven ETC activity Figure 2: Typical FAO Assay Workflow P5

7 P6 PERFORMING THE MITOXPRESS FAO ASSAY NOTE: Prior to using the MitoXpress FAO kit, ensure that measurement settings and cell number have been optimised as per the MitoXpress Xtra - Oxygen Consumption Assay User Manual. CELL CULTURE AND PLATING Count cells and adjust to the desired plating density in culture medium, (typically ~ cells per well for 2D cultures). Return the plate to culture overnight (typically >14 h). See Appendix A Explanatory Notes for more information on the use of 3D cultures and suspension cells. PRE-ASSAY PREPERATION Prepare Kit Components: - L-Carnitine: To prepare 50 mm stock dissolve vial contents in 400 µl (100X) of sterile distilled water. Aliquot in 100 µl volumes and store at -20 C, each aliquot sufficient for 100 wells (0.5 mm). - FCCP: To prepare 250 µm stock dissolve vial contents in 60 µl (100X) of DMSO. Aliquot in 20 µl volumes and store at -20 C, each aliquot sufficient for 20 wells (2.5 µm). - Etomoxir: To prepare 400 µm stock, dissolve vial contents in 550 µl (10X) of sterile distilled water. Aliquot in 50 or 100 µl volumes and store at -20 C, each 100 µl aliquot sufficient for 10 wells (40 µm). - Base Glucose Deprivation Media (optional): Glucose-free DMEM (Sigma Cat# D5030), 1 mm glucose, 1.0 mm L-glutamine, 1% FBS, 100 U/mL /0.1 mg/ml Pen/Strep. Media can be stored for 2 weeks at +4 C. - Base Measurement Media: Dissolve kit buffer tablet in 100 ml of distilled water, warm to 37 C, bring to ph 7.4 using HCL and NaOH and filter sterilise. Media can be stored for 3 weeks at 4 C. NOTE: Aliquots stored at -20 C should be used within three months (avoid freeze thaw). Prepare on Day of Measurements: - Glucose Deprivation Media (optional): To Base Glucose Deprivation Media, add 0.5mM L-Carnitine (1/100 of stock). See Appendix A Explanatory Notes for more information. FA-Free Measurement Media: To Base Measurement Media, add 0.5 mm L-Carnitine (1/100 dilution - of stock), 2.5 mm glucose. Appropriate L-Carnitine, and glucose concentration can be cell-type specific and may require additional optimisation. NOTE: If long term measurements are being performed outside 5% CO 2 a HEPES supplement is recommended. FA Measurement Media: FA-free Measurement Media µμ Oleate (1/20 of 3 mm stock). - MitoXpress Xtra reagent prepared as recommended in the MitoXpress Xtra - Oxygen Consumption Assay User Manual (MX-200).

8 GLUCOSE DEPRIVATION STEP (OPTIONAL) Wash cells twice with Base Glucose Deprivation Media, add 200 µl of Glucose Deprivation Media and return to culture, typically overnight. NOTE: The optimal incubation time duration can vary depending on cell type and may require optimisation. See Appendix A Explanatory Notes for more information TYPICAL ASSAY NOTE: We recommend the use of triplicate wells for each treatment. STEP 1: Wash cells by placing the plate on a plate block heater set to assay temperature (typically 37 C) and remove spent culture media using an aspirator, being careful not to dislodge cells from the base of the wells. Using a multichannel or repeater pipette, add 100 µl of the prewarmed FA-Free Media to each well. Repeat wash step (1x). STEP 2: Add 90 µl of pre-warmed FA Measurement Media to each well except those wells being used as FA-Free controls. To these add 85 µl of FA-Free Measurement Media and 5 µl of BSA control. NOTE: FA- Free Measurement Media is used as a control to measure O2 consumption without exogenous Oleate-BSA. BSA control is added to ensure that the free concentrations of test compounds are consistent between FA and FA-Free conditions. The BSA concentration used in FAfree control wells should be consistent with the BSA concentration in samples containing Oleate-BSA (Oleate-BSA is a 2:1 conjugate). Figure 3: Washing cells STEP 3: Add 10 µl of reconstituted MitoXpress Xtra reagent to each well, except those wells for use as Blank Controls (H11 and H12, Fig. 5). Add 10 µl of FA Measurement Media to Blank Control wells. Wells H1- H10 contain MitoXpress Xtra reagent but no cells and function as a Signal Control. NOTE: If measuring a full 96-well plate, we recommend combining Steps 1 and 2 by diluting reconstituted MitoXpress Xtra stock 1 in 10 in the relevant measurement media and, using a multichannel pipette, to add 100 µl to each well. Add 100 µl of FA Measurement Media (no MitoXpress Xtra) to the Blank Control wells. Figure 4: Adding Measurement Media Figure 5: Plate Map P7

9 STEP 4: The MitoXpress FAO Assay can be used for metabolic characterisation or to determine the impact of cell treatment (drug treatment, signaling pathway modulation, genetic manipulation etc). METABOLIC CHARACTERISATION: Cellular dependence on, or preference for FAO can be determined using kit control compounds Etomoxir and FCCP in the presence and absence of added Oleate. For compound addition, add 10 µl of Etomoxir and 1 µl FCCP stocks to designated wells. Etomoxir blocks LCFA uptake, while FCCP increases cellular energy demand thereby increasing FAO dependence. See Analysis section for more details. NOTE: Ensure a minimum of 10 min between Etomoxir addition and assay measurement. CELL TREATMENT: Various cell treatments can be performed including genetic manipulation, signaling pathway modulation and drug treatment. Drug screening is used here as an exemplar application: Add test compound or vehicle (typically 1-5 µl) to test wells. Measurement is typically performed both in the presence and absence of Oleate-BSA. Additional BSA control stock is added to wells without Oleate (see step 2). 6-8 point compound dilutions are typically used. Cells are co-treated with FCCP if impact on maximal FAO is being determined. Etomoxir is used as a control. See Analysis section for more details. NOTE: We recommend keeping volume of added compound low to minimize any potential vehicle effects. Figure 6: Measure Plate STEP 5: Seal each well with 100 µl of pre-warmed HS Mineral Oil, taking care to avoid bubbles. Read the plate immediately in a fluorescence plate reader as described in the MitoXpress Xtra - Oxygen Consumption Assay User Manual. NOTE: See MitoXpress Xtra - Oxygen Consumption Assay User Manual for additional details and tips on instrument settings, oil addition and data analysis. OPTIONAL CONTROLS: Negative Biological Controls: To 2 wells containing cells, add 1 µl of an Antimycin A stock solution (100 µm in DMSO). Antimycin blocks the ETC thereby inhibiting ETC-related oxygen consumption. Coupling Controls: To 2 wells containing cells, add 1 µl of an Oligomycin stock solution (100 µm in DMSO). Oligomycin blocks the Fo/F1 ATPase highlighting the portion of oxygen consumption driving aerobic ATP production. Remaining oxygen consumption is typically due to uncoupled mitochondria. P8

10 ANALYSIS NOTE: Data processing is described in the MitoXpress Xtra - Oxygen Consumption Assay User Manual Sample FAO-Driven Oxygen Consumption Untreated cells show a steady MitoXpress Xtra signal increase reflecting ETC-driven oxygen consumption Signal Control shows probe signal in the absence of cell respiration Etomoxir treatment prevents Oleate import, resulting in reduced availability of reducing equivalents and a resultant decrease in ETC activity. The remaining ETC activity (difference between Etoxomir treatment and Signal Control) is driven by metabolic activity other than LC FAO. FCCP treatment induces maximal ETC activity by dissipating the mitochondrial membrane potential. Increased demand for reducing equivalents causes a concomitant increases in FAO as indicated by the rapid increase in MitoXpress Xtra signal. This strong increase in ETC activity is not observed where exogenous LCFA is unavailable or where import is inhibited. Evaluating Exogenous and Endogenous FAO Figure 7: FAO-driven respiration of HepG2 cells treated with the CPT-1 inhibitor Etomoxir and uncoupler FCCP. FAO-driven respiratory activity can be interrogated further by calculating the rate of signal change for each MitoXpress Xtra FAO assay profile, facilitating assessment of exogenous FAO (Oleate supplied), endogenous FAO (Oleate-free) and non-lc FAO, (Etomoxir treated). This can be determined using slopes (m) calculated from the linear portion of each profile: - Exogenous FAO = m Oleate - m Etomoxir - Endogenous FAO = moleate-free - metomoxir - Non-LC FAO = m Etomoxir m Signal Control Sample data (right) summarises the balance between these parameters under Basal and Maximum (FCCP treated) conditions and clearly illustrates that the increased energy demand imposed by FCCP treatment is met by increased FAO. Figure 8: FAO-driven respiration of HepG2 cells treated with the CPT-1 inhibitor Etomoxir and uncoupler FCCP. P9

11 ANALYSIS NOTE: Data processing is described in the MitoXpress -Xtra O2 consumption user manual (p11). Metabolic Characterisation As described in Figure 8, the MitoXpress FAO kit facilitates interrogation of oxygen consumption due to exogenous FA substrates such as Oleate, endogenous FA stores, and non long chain FA substrates. Sample data (right) uses this approach to determine the impact of differentiation on C2C12 cell metabolism, measuring maximal respiratory capacity of fully confluent C2C12 myoblasts (undifferentiated) and multinucleated myotubes (differentiated), and the proportion of this capacity driven by long chain FAO. Differentiation from myoblasts to multinucleated myotubes significantly increases maximal respiratory capacity, measured after a 1h glucose deprivation step. This is driven by both endogenous substrates and an increased capacity to metabolise exogenous FA substrates (Oleate-BSA) to meet an increased demand for reducing equivalents imposed by FCCP treatment. Figure 9: Maximal FAO-driven respiration of differentiated and undifferentiated C2C12 cells (FCCP treated). (Data courtesy of Dr Ben Buehrer, Zen-Bio Inc.) P10

12 APPENDIX A - EXPLANATORY NOTES MitoXpress Xtra - Oxygen Consumption Assay The MitoXpress FAO is designed as a Companion Kit for use in combination with Luxcel s MitoXpress Xtra - Oxygen Consumption Assay (MX-200). The Oxygen Consumption Assay User Manual describes instrument set-up, assay optimisation, data analysis and troubleshooting. The described instrument set-up and signal optimisation steps should be performed prior running a MitoXpress FAO assay. Cell Plating Typically cells are seeded at a density to achieve full confluence on the day of measurement. Plating density, cell type and basal metabolic rate will determine oxygen consumption rate. When plating 3D cultures prepare the plate or matrix solution in advance as per the manufacturer s instructions. Cells are typically plated at higher concentrations for 3D cultures. If using extended culture periods (>2 days), for best results, plate as per recommended plate map (Fig. 10) adding 200 µl of culture medium to all outer wells. This minimizes plate effects related to inconsistent cell growth across the microplate. For some cell types, inconsistent growth can be additionally reduced by allowing the plate to stand Figure 10: Recommended plate map for extended culture times (> 2 days) at room temperature for 30 min prior to being returned to culture [5]. Test compounds can take longer to penetrate and effect 3D cultures and longer treatment times can be considered in these instances. Glucose Deprivation A glucose deprivation step can be included prior to measurement to increase cellular dependence on FAO. Typical Glucose Deprivation Media formulation and duration are described, however optimum conditions and duration are cell-type dependent. For maximum FAO dependence, L-Carnitine, glucose and Oleate concentration should be optimised and the most effective glucose deprivation duration determined. If an overnight glucose deprivation step to be included with non-terminally differentiated cells, seeding densities should be adjusted downwards to facilitate doubling. Long-Term Measurement without CO 2 control Additional Media buffering capacity is required when conducting long term measurements (>2h) outside 5% CO 2. This is achieved by supplementing Measurement Media with 5 mm HEPES. Supplementation is not required if the plate reader being used has environmental gas control where 5% CO 2 can be maintained within the measurement chamber. P11

13 Optimising concentrations and treatment times for assay controls FCCP exhibits a bell-shaped dose-response which can vary between cell types. The concentration which delivers maximum respiratory activity should be determined for each cell type. This can be achieved by running an FCCP serial dilution (typically between µm) in the presence of Oleate-BSA. Higher FCCP concentrations may be required when using Oleate-BSA conjugates as compared with glucose-based measurement due to the ability of BSA to bind FCCP. Oleate-BSA is a 2:1 conjugate. Oleate-BSA is typically used at 150 µm, however the concentration at which maximum respiratory activity is observed can be cell type dependent. Optimum concentration can be determined by measuring oxygen consumption at varying Oleate-BSA concentrations (typically µm) in the presence of FCCP. Users may also wish to add Oleate-BSA to Glucose Deprivation Media, (typically 100 µm). L-Carnitine is typically used at 0.5 mm, however the optimum concentration to facilitate LCFA transport can be cell type dependent. Optimum concentration can be determined by measuring oxygen consumption at varying L-Carnitine concentrations in the presence of FCCP. Etomoxir can exhibit off-target effects at concentrations above 40 µm, however efficacy can be reduced at elevated serum and BSA concentrations. In these instances, higher Etomoxir concentrations can be used to ensure CPT-1 inhibition. A minimum of 10 minutes should elapse between Etomoxir treatment and the commencement of measurement to ensure CPT-1 inhibition has impacted oxygen consumption prior to measurement. To maximize inhibition, Etomoxir can be pre-incubated in FA-Free Media prior to the addition of Oleate-BSA or BSA control. Compounds are typically added immediately pre-treatment to determine their effect on FAO and related mitochondrial function. For some 3D models, a pre-incubation step can be incorporated to ensure compounds access cells within the 3D construct. Longer treatment times can be used as required; in these instances compound should be present in both culture media (during incubation) and measurement media. P12

14 REFERENCES [1] Mammalian mitochondrial beta-oxidation. Eaton et al, Biochem J., 1996; 320: [2] Regulation of Fatty Acid Oxidation in Heart. Schulz et al., J. Nutr., 1994; 124: [3] Cancer metabolism: fatty acid oxidation in the limelight. Carracedo et al., 2013; Nat Rev Cancer, 4: [4] Drug-induced toxicity on mitochondria and lipid metabolism: mechanistic diversity and deleterious consequences for the liver. Begriche et al., J Hepatol., 2011; 54: , [5] A Simple Technique for Reducing Edge Effect in Cell-Based Assays. Lundholt et al., J. Biomol. Screen. 2003; 5: P13

15 RELATED PRODUCTS MitoXpress Xtra Oxygen Consumption Assay [HS Method] (Cat No. MX-200) MitoXpress Intra Intracellular Oxygen Assay (Cat No. MX-300)

16 UM-04 V.1.2