BAM-SLU Melt 🔥 Blend

Research Overview

BAM-SLU MELT Blend serves as an advanced research tool for investigating mitochondrial uncoupling mechanisms and their synergistic effects on cellular metabolism in laboratory settings. Mitochondrial uncouplers dissociate oxidative phosphorylation from ATP synthesis by increasing proton leak across the inner mitochondrial membrane, resulting in increased oxygen consumption, substrate oxidation, and heat production (thermogenesis) without proportional ATP generation. This fundamental metabolic process represents a critical area of investigation for obesity, diabetes, metabolic syndrome, aging, and cellular energy homeostasis research.

Unlike classical single-compound approaches, the BAM-SLU MELT Blend combines two structurally distinct mitochondrial uncouplers with complementary characteristics, enabling investigation of multiple research questions simultaneously. BAM-15 represents a next-generation mitochondrial uncoupler with reported improved selectivity for mitochondrial membranes over plasma membranes, potentially reducing off-target effects associated with classical uncouplers. SLU-PP-332 demonstrates milder uncoupling activity with a more gradual dose-response relationship, allowing investigation of subtle metabolic shifts without severe mitochondrial dysfunction.

The combination approach offers researchers several unique advantages: (1) investigation of synergistic uncoupling effects, (2) comparison of distinct molecular mechanisms within the same experimental system, (3) potential for enhanced metabolic effects at lower individual compound concentrations, (4) examination of complementary pathway activation, and (5) modeling of complex metabolic interventions. Research applications span mitochondrial bioenergetics, metabolic disease models, thermogenesis research, obesity and diabetes investigations, aging and longevity studies, cellular stress response pathways, and neuroscience applications.

Laboratory studies examine the blend’s effects on mitochondrial respiration, membrane potential, ATP production, substrate utilization, thermogenesis, energy expenditure, gene expression, metabolic pathway activation, and whole-organism metabolic parameters in cell culture systems, isolated mitochondria, tissue explants, and preclinical animal models. The dual-component formulation enables comprehensive investigation of mitochondrial uncoupling biology while maintaining experimental efficiency.

Component Analysis: BAM-15

Molecular Characteristics:

• CAS Number: 1803860-99-0
• Molecular Weight: 500.6 Da
• Molecular Formula: C₂₈H₂₇ClFN₃O₃
• Chemical Class: Synthetic mitochondrial uncoupler (protonophore)
• Appearance: White to off-white powder
• Solubility: DMSO, ethanol (organic solvents), limited aqueous solubility

BAM-15 Mechanism and Characteristics:

BAM-15 represents a designed mitochondrial uncoupler with reported improved selectivity profile compared to classical uncouplers such as 2,4-dinitrophenol (DNP) or carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP). The compound functions as a lipophilic weak acid (protonophore) that shuttles protons across biological membranes, dissipating the proton-motive force that normally drives ATP synthesis.

Research characterizing BAM-15 has identified several distinguishing features:

Mitochondrial Selectivity: BAM-15 demonstrates preferential uncoupling of mitochondrial membranes relative to plasma membranes. Classical uncouplers like DNP can dissipate proton gradients across multiple cellular membranes, potentially causing toxicity through disruption of plasma membrane potential and cellular ion homeostasis. BAM-15’s reported selectivity reduces these off-target effects, making it potentially more suitable for chronic treatment studies and in vivo research applications.

Dose-Response Profile: The compound exhibits a dose-dependent increase in oxygen consumption and mitochondrial respiration across a range of concentrations. Research suggests a relatively broad concentration range showing uncoupling effects without immediate cytotoxicity, allowing investigation of varying degrees of metabolic stimulation.

Metabolic Effects: BAM-15 research has documented increased oxygen consumption, enhanced fatty acid oxidation, elevated thermogenesis, improved glucose homeostasis, reduced body weight gain in diet-induced obesity models, and improved metabolic parameters without apparent toxicity at research doses. These effects make BAM-15 valuable for obesity research, diabetes research, and metabolic disease investigations.

Mechanistic Basis: As a protonophore, BAM-15 increases proton leak across the inner mitochondrial membrane. The lipophilic neutral form diffuses across the membrane from the intermembrane space into the matrix, where it releases its proton. The charged deprotonated form then returns across the membrane to repeat the cycle. This futile cycling dissipates the proton gradient, reducing the proton-motive force available for ATP synthesis and converting the energy into heat instead.

The molecular structure of BAM-15 contains functional groups enabling proton shuttling while maintaining structural characteristics that favor mitochondrial membrane partitioning over plasma membrane interactions. The specific combination of lipophilicity, pKa characteristics, and molecular architecture contributes to its selectivity profile.

Component Analysis: SLU-PP-332

Molecular Characteristics:

• Molecular Weight: 456.5 Da
• Molecular Formula: C₂₄H₂₈N₄O₅
• Chemical Class: Synthetic mitochondrial uncoupler (mild, selective)
• Appearance: Powder
• Solubility: DMSO, ethanol (organic solvents), limited aqueous solubility

SLU-PP-332 Mechanism and Characteristics:

SLU-PP-332 represents another next-generation mitochondrial uncoupler with distinct characteristics from classical uncouplers and from BAM-15. The compound demonstrates milder uncoupling activity with a more gradual concentration-response relationship, enabling investigation of subtle metabolic effects without the severe mitochondrial dysfunction associated with high-potency uncouplers.

Research characterizing SLU-PP-332 has identified several key features:

Mild Uncoupling Profile: SLU-PP-332 increases mitochondrial respiration and oxygen consumption in a dose-dependent manner, but with less dramatic effects on mitochondrial membrane potential at lower concentrations compared to classical uncouplers. This mild uncoupling characteristic allows investigation of metabolic stimulation that may more closely resemble physiological uncoupling processes (such as those mediated by uncoupling proteins like UCP1 in brown adipose tissue).

Gradual Dose-Response: Unlike classical uncouplers that often show steep dose-response curves with narrow therapeutic windows, SLU-PP-332 exhibits a more gradual concentration-response relationship. This property provides a wider experimental window for investigating varying degrees of metabolic stimulation and reduces the risk of excessive uncoupling-induced toxicity.

Metabolic Effects: Research with SLU-PP-332 has examined effects on oxygen consumption, substrate utilization, thermogenesis, metabolic pathway activation (particularly AMPK signaling), mitochondrial biogenesis, and metabolic parameters in obesity and diabetes models. The milder activity profile makes SLU-PP-332 particularly suitable for chronic treatment studies and investigations of adaptive metabolic responses.

Cellular Tolerability: The milder uncoupling activity generally results in better cellular tolerability in culture systems and potentially improved safety profiles in preclinical models. This characteristic enables longer-duration studies and investigation of chronic metabolic adaptation.

Mechanistic Basis: SLU-PP-332 functions as a mitochondrial uncoupler through proton shuttling mechanisms similar to other chemical uncouplers, but the specific molecular characteristics result in its distinctive mild uncoupling profile. The compound increases proton leak across the inner mitochondrial membrane, dissipating the proton-motive force and converting energy to heat rather than ATP.

The molecular structure of SLU-PP-332 contains functional groups enabling membrane partitioning and proton binding/release, but the specific structural features result in milder activity compared to high-potency uncouplers. This may reflect factors such as pKa values, lipophilicity, membrane partitioning kinetics, or proton transfer rates that differ from classical uncouplers.

Synergistic Mechanisms and Research Rationale

The combination of BAM-15 and SLU-PP-332 in the MELT Blend provides unique research opportunities for investigating synergistic and complementary uncoupling mechanisms:

Complementary Activity Profiles:
BAM-15’s mitochondrial selectivity combined with SLU-PP-332’s mild uncoupling characteristics may provide enhanced metabolic stimulation with reduced toxicity risk. The combination enables investigation of whether two distinct uncoupling mechanisms produce additive or synergistic effects on metabolic parameters.

Multiple Mechanism Investigation:
Researchers can examine how different uncoupling mechanisms interact within the same experimental system. This approach provides insights into the fundamental biology of mitochondrial uncoupling and energy homeostasis that single-compound studies cannot address.

Dose Optimization:
The combination may enable achievement of desired metabolic effects at lower concentrations of each individual component, potentially reducing concentration-dependent toxicity while maintaining or enhancing efficacy. This research question is particularly relevant for translational studies.

Pathway Activation Comparison:
Different uncoupling compounds may activate distinct downstream signaling pathways or show varying effects on metabolic sensors (AMPK, mTOR, sirtuins). The blend enables comparative investigation of pathway activation patterns and identification of compound-specific versus class effects.

Metabolic Phenotype Investigation:
The combination may produce distinct metabolic phenotypes (substrate preference, respiratory patterns, thermogenic capacity) compared to either compound alone, providing insights into metabolic flexibility and adaptation mechanisms.

Temporal Response Patterns:
BAM-15 and SLU-PP-332 may show different pharmacokinetic profiles or temporal response patterns. The combination enables investigation of sustained metabolic effects that may result from complementary temporal dynamics.

Research Applications

Mitochondrial Bioenergetics Research

The BAM-SLU MELT Blend enables comprehensive investigation of mitochondrial uncoupling mechanisms and bioenergetic principles:

Oxygen Consumption and Respiration Studies:

• Seahorse XF analyzer measurements of oxygen consumption rate (OCR) with real-time assessment
• Basal respiration, ATP-linked respiration, maximal respiratory capacity, and spare respiratory capacity quantification
• Dose-response characterization for each component and the combination
• Comparison with classical uncouplers (DNP, FCCP) and vehicle controls
• Substrate-specific respiration (complex I substrates, complex II substrates, fatty acid oxidation)
• Coupling efficiency calculations (ATP production rate / oxygen consumption rate)

Mitochondrial Membrane Potential Measurements:

• Fluorescent probe-based measurements (TMRM, JC-1, rhodamine 123)
• Flow cytometry analysis of mitochondrial polarization across cell populations
• Live-cell confocal microscopy of membrane potential dynamics
• Concentration-response relationships for membrane potential changes
• Assessment of synergistic depolarization effects
• Correlation of membrane potential changes with functional outcomes

ATP Production and Energy Charge Analysis:

• Cellular ATP content measurement using luciferase-based bioluminescence assays
• ATP/ADP and ATP/AMP ratio determination by HPLC or mass spectrometry
• Real-time ATP production rate assessment
• P/O ratio calculations (ATP produced per oxygen consumed)
• Adenylate energy charge [(ATP + 0.5ADP)/(ATP + ADP + AMP)]
• Effects on cellular energy status and metabolic compensation

Proton Leak Quantification:

• Direct measurement of proton leak rate in isolated mitochondria
• Kinetic analysis of leak-dependent respiration
• Quantification of uncoupling efficiency
• Comparison of leak rates induced by each component and the combination
• Correlation with structural features and concentration

Mitochondrial Dynamics and Morphology:

• Live-cell imaging of mitochondrial network structure
• Quantification of mitochondrial length, branching, connectivity
• Assessment of fusion and fission dynamics (DRP1, MFN1/2, OPA1 markers)
• Effects of metabolic stress on mitochondrial morphology
• Relationship between bioenergetic state and structural organization
• Mitophagy induction and mitochondrial turnover

Research protocols employ multiple experimental systems including isolated mitochondria (highest resolution of direct effects), permeabilized cells (maintain cytosolic factors), intact cells (physiological context), and tissue preparations (organ-specific characteristics) to characterize effects across biological complexity levels.

Metabolic Research Applications

The MELT Blend enables comprehensive investigation of metabolic regulation and energy balance:

Substrate Utilization and Metabolic Flux:

• Glucose oxidation measurements using radiolabeled glucose (¹⁴C-glucose)
• Glycolytic rate assessment (extracellular acidification rate by Seahorse XF)
• Fatty acid oxidation using radiolabeled palmitate or oleate
• Ketone body production and utilization
• Amino acid catabolism and nitrogen metabolism
• Substrate preference analysis under various nutrient conditions
• Metabolic flexibility testing (ability to switch between substrates)
• Nutrient sensor activation (AMPK phosphorylation, mTOR activity, SIRT1/3 activation)

Thermogenesis Research:

• Direct heat production measurement using isothermal calorimetry
• Indirect calorimetry (oxygen consumption and CO₂ production)
• Brown adipose tissue (BAT) activation studies in rodent models
• UCP1-dependent and UCP1-independent thermogenesis investigation
• Cold-induced thermogenesis protocols
• Diet-induced thermogenesis (thermic effect of feeding)
• Body temperature measurements (core and peripheral)
• Thermal imaging of BAT depots

Energy Expenditure Analysis:

• Comprehensive metabolic phenotyping using metabolic cages (CLAMS, TSE systems)
• 24-hour oxygen consumption and CO₂ production profiles
• Respiratory exchange ratio (RER = VCO₂/VO₂) determination for fuel preference
• Activity-corrected energy expenditure (separating movement from metabolic effects)
• Resting metabolic rate and total energy expenditure
• Feed efficiency calculations
• Energy balance studies (intake vs. expenditure)

Body Composition and Weight Management:

• Body weight monitoring in diet-induced obesity models
• Fat mass and lean mass quantification (NMR, DEXA scanning)
• Adipose tissue depot weights (visceral, subcutaneous, brown adipose tissue)
• Liver lipid content and hepatic steatosis assessment
• Effects on weight gain prevention versus weight loss
• Comparison with caloric restriction and exercise interventions

Metabolic Disease Models:

• High-fat diet-induced obesity models
• High-fat high-sucrose diet models for metabolic syndrome
• Genetic obesity models (ob/ob, db/db, diet-induced obese-resistant strains)
• Type 2 diabetes models (db/db, ZDF rats, diet-induced insulin resistance)
• Non-alcoholic fatty liver disease (NAFLD) and NASH models
• Metabolic aging models
• Lipodystrophy models

Experimental approaches integrate cell culture models (adipocytes, hepatocytes, myocytes, pancreatic beta cells), tissue explants, and rodent models with comprehensive metabolic phenotyping platforms to characterize systemic metabolic effects.

Obesity and Diabetes Research

Mitochondrial uncoupling represents a promising research approach for metabolic disease based on the principle of increasing energy expenditure and substrate oxidation:

Adipose Tissue Research:

• White adipose tissue lipolysis and lipid mobilization
• Fatty acid oxidation in white adipocytes
• Brown adipose tissue thermogenic capacity and UCP1 expression
• Beige/brite adipocyte induction in subcutaneous white adipose tissue
• Adipose tissue gene expression profiling (UCP1, PGC-1α, PRDM16, PPARγ, PPARα)
• Adipokine secretion profiles (leptin, adiponectin, resistin)
• Adipose tissue inflammation and macrophage infiltration
• Mitochondrial content and function in adipocytes

Hepatic Metabolism:

• Hepatic glucose production and gluconeogenesis regulation
• Fatty acid synthesis (de novo lipogenesis) suppression
• Fatty acid oxidation enhancement
• Triglyceride accumulation and lipid droplet dynamics
• Hepatic insulin sensitivity and insulin signaling
• Mitochondrial function in NAFLD/NASH models
• Hepatic inflammation and fibrosis markers
• Liver enzyme profiles

Skeletal Muscle Metabolism:

• Muscle glucose uptake and insulin-stimulated glucose disposal
• GLUT4 translocation and insulin signaling pathways
• Mitochondrial biogenesis and oxidative capacity
• Exercise-mimetic effects and endurance capacity
• Muscle fiber type characteristics (oxidative vs. glycolytic)
• Myokine secretion (irisin, IL-6, IL-15)
• Lipid accumulation in muscle (intramyocellular lipid)
• Muscle protein synthesis and degradation balance

Pancreatic Beta Cell Function:

• Glucose-stimulated insulin secretion
• Beta cell mass and proliferation
• Effects of improved metabolic status on beta cell health
• Protection against lipotoxicity and glucotoxicity
• Beta cell mitochondrial function

Whole-Body Glucose Homeostasis:

• Oral and intraperitoneal glucose tolerance tests (OGTT, IPGTT)
• Insulin tolerance tests (ITT)
• Hyperinsulinemic-euglycemic clamp studies (gold standard)
• Fasting and postprandial glucose and insulin profiles
• HbA1c and fructosamine measurements for long-term glycemic control
• Glucose uptake in specific tissues using radiolabeled glucose
• Hepatic and peripheral insulin sensitivity quantification

Research investigates whether dual mitochondrial uncoupling via the MELT Blend produces enhanced metabolic improvements compared to single compounds, potentially through synergistic effects on multiple tissue types or metabolic pathways.

Cellular Stress Response and Adaptive Signaling

Mitochondrial uncoupling activates cellular stress response pathways that may mediate long-term adaptive benefits:

AMPK Activation and Signaling:

• AMPK phosphorylation at Thr172 (activating phosphorylation)
• Upstream kinase activation (LKB1, CaMKK2)
• Downstream target phosphorylation (ACC, TBC1D1, TSC2, ULK1)
• AMPK-dependent gene expression changes
• Metabolic consequences (glucose uptake, fatty acid oxidation, protein synthesis inhibition)
• Comparison with pharmacological AMPK activators (AICAR, metformin, A-769662)
• Time course of AMPK activation (acute vs. chronic effects)

Mitochondrial Biogenesis:

• PGC-1α expression and activity (master regulator of mitochondrial biogenesis)
• NRF1 and NRF2 (nuclear respiratory factors) activation
• TFAM (mitochondrial transcription factor A) expression
• Mitochondrial DNA copy number quantification
• Mitochondrial protein expression (OXPHOS complexes I-V)
• Citrate synthase and cytochrome c oxidase enzyme activities
• Mitochondrial mass measurements (MitoTracker, citrate synthase, mitochondrial protein content)
• Time course of biogenic response (transcription, translation, organelle assembly)

Mitochondrial Quality Control:

• Mitophagy induction and flux measurements
• PINK1-Parkin pathway activation
• LC3 lipidation and autophagosome formation
• Mitochondrial protein import efficiency
• Mitochondrial unfolded protein response (UPRmt) activation
• Proteolytic pathways (Lon protease, ClpP, matrix proteases)
• Segregation of damaged mitochondria
• Balance of biogenesis and degradation (mitochondrial turnover)

Oxidative Stress and Antioxidant Responses:

• Reactive oxygen species (ROS) production measurement (DHE, MitoSOX, H2DCFDA)
• Superoxide, hydrogen peroxide, and hydroxyl radical quantification
• Antioxidant enzyme expression (SOD1/2, catalase, GPx, peroxiredoxins)
• Glutathione levels (reduced GSH, oxidized GSSG, GSH/GSSG ratio)
• NADPH availability
• Lipid peroxidation markers (MDA, 4-HNE)
• Protein carbonylation and oxidative damage
• Nrf2 pathway activation and antioxidant response element (ARE) transcription

Metabolic Gene Expression:

• RNA sequencing or microarray analysis of global transcriptional changes
• Metabolic pathway gene expression (glycolysis, TCA cycle, fatty acid oxidation, gluconeogenesis)
• Transcription factor activation (PPARs, PGC-1α, FOXO, HIF-1α)
• Nuclear-mitochondrial communication pathways
• Epigenetic modifications affecting metabolic gene expression

Research examines how dual uncoupling activates adaptive pathways that may confer long-term metabolic benefits, potentially through enhanced or complementary signaling compared to single compounds.

Aging and Longevity Research

Mitochondrial function plays central roles in aging processes, and mild metabolic stress may activate longevity pathways:

Mitochondrial Aging:

• Age-related decline in mitochondrial function and oxidative capacity
• Mitochondrial DNA mutations, deletions, and damage accumulation
• Oxidative damage to mitochondrial proteins and lipids
• Mitochondrial-derived reactive oxygen species (mtROS) and aging
• Changes in mitochondrial dynamics and morphology with aging
• Decline in mitochondrial quality control mechanisms
• Effects of mild uncoupling on mitochondrial aging parameters
• Potential rejuvenation of aged mitochondria

Healthspan and Lifespan Studies:

• Longevity assessments in model organisms (C. elegans, Drosophila, mice)
• Median and maximal lifespan determination
• Age-related disease onset and progression (cancer, neurodegeneration, cardiovascular disease)
• Physical performance and frailty measurements (grip strength, rotarod, treadmill endurance)
• Cognitive function assessments (Morris water maze, novel object recognition)
• Inflammatory markers and immunosenescence
• Maintenance of tissue function with aging
• Compression of morbidity (extending healthy years)

Caloric Restriction Mimetics:

• Molecular similarities between mild uncoupling and caloric restriction
• Overlapping pathways (AMPK activation, SIRT1/3 activation, mTOR inhibition)
• NAD+ metabolism and sirtuin activation
• FOXO transcription factor activation
• Metabolic reprogramming and substrate switching
• Stress resistance and hormesis
• Comparison of metabolic, transcriptional, and physiological effects
• Potential for capturing caloric restriction benefits without dietary restriction

Cellular Senescence:

• Effects on senescent cell accumulation
• Senescence-associated secretory phenotype (SASP)
• Mitochondrial dysfunction in senescent cells
• Potential senomorphic effects (modulating senescent cell function)
• Interaction with senolytics

Research investigates whether dual mitochondrial uncoupling activates longevity pathways more effectively than single compounds, potentially through enhanced metabolic stress responses or complementary pathway activation.

Neuroscience Research Applications

Mitochondrial function profoundly influences neuronal health, synaptic function, and neurodegenerative processes:

Neuronal Bioenergetics:

• Neuronal ATP production and energy status
• Synaptic transmission energy requirements (ion pumping, vesicle recycling)
• Axonal transport energy dependence and mitochondrial trafficking
• Dendritic spine energetics
• Effects of mild uncoupling on neuronal function and viability
• Neuroprotection versus neurotoxicity concentration thresholds
• Regional brain metabolic differences
• Neuron-astrocyte metabolic coupling (lactate shuttle)

Neurodegenerative Disease Models:

• Parkinson’s disease models (MPTP, 6-OHDA, rotenone, genetic models with mitochondrial complex I deficiency)
• Alzheimer’s disease models (APP/PS1, 3xTg, amyloid beta oligomer effects on mitochondria)
• Huntington’s disease models (mutant huntingtin mitochondrial effects, striatal vulnerability)
• Amyotrophic lateral sclerosis (ALS) models (SOD1 mutations, mitochondrial impairment in motor neurons)
• Mitochondrial dysfunction as common feature across neurodegenerative diseases
• Potential neuroprotective effects of mild metabolic enhancement
• Effects on protein aggregation and clearance
• Synaptic protection and preservation of neuronal connectivity

Neuroinflammation:

• Microglial metabolic phenotypes (M1 pro-inflammatory vs. M2 anti-inflammatory)
• Inflammatory mediator production (cytokines, chemokines, prostaglandins)
• Effects of metabolic modulation on neuroinflammation
• Blood-brain barrier integrity and permeability
• Astrocyte activation and reactive gliosis
• Mitochondrial effects on inflammatory signaling (NLRP3 inflammasome, NF-κB)

Cognitive Function:

• Learning and memory assessments in rodent models
• Synaptic plasticity (long-term potentiation, long-term depression)
• Neurogenesis in hippocampus
• Effects on age-related cognitive decline
• Potential cognitive enhancement through metabolic optimization

Blood-Brain Barrier Penetration:

• Assessment of compound brain penetration
• Brain tissue pharmacokinetics
• Effects on central versus peripheral metabolism
• Regional brain distribution

Research examines whether mild mitochondrial uncoupling improves neuronal resilience, provides neuroprotection in disease models, or enhances cognitive function, and whether the dual-compound approach offers advantages over single compounds.

Laboratory Handling and Storage Protocols

Powder Storage:

• Store at -20°C in sealed container with desiccant
• Protect from light exposure (amber container or foil wrap)
• Maintain desiccated environment to prevent moisture absorption
• Minimize opening frequency to limit moisture exposure
• Record receipt date and first opening date
• Each component maintains separate stability under these conditions
• Stability data available for recommended storage conditions

Stock Solution Preparation:

• Dissolve in DMSO (primary recommended solvent) to create concentrated stock solutions
• Typical stock concentrations: 10-100 mM depending on solubility limits of each component
• Add DMSO slowly and vortex thoroughly to ensure complete dissolution
• Visual inspection for complete dissolution (clear solution with no particulates)
• May require gentle warming (37°C water bath, maximum 5-10 minutes) or brief sonication
• Never use direct heat or open flame
• Verify concentration by UV spectroscopy if critical for experiments
• Document stock concentration, preparation date, solvent, lot number for traceability

Stock Solution Storage:

• Aliquot immediately after preparation into single-use volumes (50-200 μL)
• Use polypropylene tubes (not polystyrene – some compounds bind)
• Store at -20°C for long-term storage
• Protect from light (amber tubes or aluminum foil wrap)
• Avoid repeated freeze-thaw cycles (maximum 2-3 cycles recommended)
• Single-use aliquots strongly recommended for consistency
• Label with compound name, concentration, solvent, preparation date, lot number
• Record thaw history if aliquots are reused

Working Solution Preparation:

• Dilute DMSO stock solutions into appropriate cell culture medium, buffer, or vehicle for animal studies
• Add DMSO stock dropwise with mixing to prevent local concentration effects
• Final DMSO concentration in cell culture: ≤0.1-0.5% (titrate for each cell type)
• For animal studies, appropriate vehicles required (PEG-400, Tween-80, hydroxypropyl-β-cyclodextrin, or combinations)
• Prepare fresh working solutions for each experiment when possible
• Filter sterilize for cell culture applications (0.22 μm syringe filter)
• Include vehicle controls at equivalent DMSO/vehicle concentration in all experiments
• Document dilution scheme for reproducibility

Handling Precautions:

• Wear appropriate personal protective equipment (lab coat, nitrile gloves, safety glasses)
• Handle in chemical fume hood or well-ventilated area
• Avoid skin contact and inhalation of powder or aerosols
• Follow institutional chemical safety protocols and SDS recommendations
• Dispose as hazardous chemical waste per institutional and local regulations
• Clean spills immediately with appropriate absorbent material
• Wash hands thoroughly after handling even when gloved
• Do not eat, drink, or apply cosmetics in areas where compounds are handled

Special Considerations for Mitochondrial Uncouplers:

• These compounds affect cellular viability at higher concentrations
• Establish concentration-response relationships in each experimental system before large studies
• Start with broad concentration ranges (e.g., 0.1-100 μM) and narrow based on initial results
• Monitor cell viability alongside metabolic measurements (MTT, ATP content, LDH release)
• Cytotoxicity thresholds vary by cell type, exposure duration, and metabolic status
• Temperature affects uncoupling activity (experiments at 37°C vs. room temperature show different effects)
• Serum in culture medium may affect compound availability through protein binding
• Consider metabolic state of cells (proliferating vs. confluent, glucose vs. fatty acid medium)

Quality Assurance and Analytical Testing

Each BAM-SLU MELT Blend batch undergoes comprehensive analytical characterization with separate analysis for each component:

Purity Analysis:

• High-Performance Liquid Chromatography (HPLC): ≥98% purity for each component
• Reversed-phase HPLC with optimized gradient and column for each compound
• UV detection at compound-specific wavelengths
• Multiple peak integration for accurate purity determination
• Impurity identification, quantification, and tracking across batches
• Verification that each component is present at specified ratio

Structural Verification:

• Electrospray Ionization Mass Spectrometry (ESI-MS): Confirms molecular weight for BAM-15 (500.6 Da) and SLU-PP-332 (456.5 Da)
• High-resolution mass spectrometry for exact mass determination
• Nuclear Magnetic Resonance (NMR): ¹H-NMR and ¹³C-NMR structural verification for each component
• Infrared spectroscopy (IR) for functional group confirmation
• Comparison with reference standards
• Verification of component identity and absence of isomeric impurities

Quantitative Composition:

• Quantification of each component by HPLC with external calibration
• Verification of blend ratio
• Total compound content determination
• Consistency verification across production batches

Contaminant Testing:

• Bacterial endotoxin: <5 EU/mg (LAL method) for cell culture applications
• Heavy metals: Below detection limits per USP and standards
• Residual solvents: Gas chromatography-mass spectrometry (GC-MS) quantification within ICH limits
• Water content: Karl Fischer titration
• Related substances and degradation products monitored by HPLC
• Sterility testing for applications requiring sterile conditions

Documentation:

• Comprehensive Certificate of Analysis (COA) provided with each batch
• Analytical chromatograms and spectra available upon request
• Third-party analytical verification available for quality confirmation
• Batch-specific QC results traceable by lot number
• Stability data for recommended storage conditions
• Chain of custody documentation

Research Considerations

Experimental Design Factors:

1. Concentration Selection: Determine appropriate concentration ranges through preliminary dose-response studies. Mitochondrial uncouplers typically show effects at μM concentrations in cell culture. Start with broad ranges (e.g., 0.1-100 μM for each component) and narrow based on initial results. Consider testing blend at various ratios to identify optimal combinations. Include concentrations of individual components for comparison.

2. Ratio Optimization: The provided blend has a defined ratio, but researchers may wish to investigate alternative ratios. This requires separate stocks of BAM-15 and SLU-PP-332. Factorial design experiments can identify synergistic concentration combinations.

3. Time Course Considerations: Mitochondrial uncoupling effects span multiple timescales. Immediate effects (seconds to minutes) include membrane depolarization and respiration increase. Acute effects (minutes to hours) include metabolic pathway activation and energy sensor responses. Subacute effects (hours to days) include transcriptional responses and protein expression changes. Chronic effects (days to weeks) include adaptive responses, mitochondrial biogenesis, and metabolic reprogramming. Design time courses appropriate for research questions and endpoints.

4. Temperature Control: Uncoupling activity is temperature-dependent due to effects on membrane fluidity, diffusion rates, and protonation equilibria. Maintain consistent temperature during measurements. Pre-warm solutions and maintain instruments at physiological temperature (37°C for mammalian systems). Consider temperature effects when extrapolating between in vitro and in vivo results.

5. Cell Type and Metabolic State: Different cell types show varying sensitivity to uncouplers based on metabolic characteristics and mitochondrial content. Highly metabolic cells (neurons, cardiomyocytes, hepatocytes, brown adipocytes) show greater sensitivity than less metabolic cells (fibroblasts, resting lymphocytes). Proliferating cells versus confluent cells show different responses. Substrate availability (glucose, fatty acids, amino acids) influences uncoupling effects.

6. Vehicle Controls: Always include vehicle controls matching DMSO or other solvent concentration in all treatment groups. DMSO itself can affect cellular metabolism at concentrations above 0.5-1%. Characterize vehicle effects in initial experiments. Include both vehicle-only controls and untreated controls when possible.

7. Positive Controls: Include well-characterized uncouplers (FCCP at 0.5-2 μM, DNP at appropriate concentrations) as positive controls to validate experimental systems, confirm mitochondrial functionality, and provide reference points for comparing potency. Include individual BAM-15 and SLU-PP-332 treatments to assess synergy.

Mechanism Validation:

Confirm mitochondrial uncoupling mechanism through multiple complementary approaches:

Direct Mitochondrial Effects (Required):

• Oxygen consumption rate increase (primary indicator)
• Mitochondrial membrane potential decrease (confirms depolarization)
• ATP production rate decrease (confirms uncoupling from ATP synthesis)
• Proton leak rate increase (direct mechanistic measurement in isolated mitochondria)
• Dose-response relationships for all parameters
• Temporal dynamics of responses

Cellular Metabolic Consequences (Supporting):

• Increased substrate oxidation (glucose, fatty acids)
• AMPK activation (responds to energy stress)
• Compensatory glycolysis upregulation (maintains ATP levels)
• Heat production increase (calorimetry)
• Nutrient sensor pathway activation

Specificity Controls (Validation):

• Effects require functional electron transport chain (blocked by rotenone, antimycin A)
• Effects persist in presence of oligomycin (ATP synthase inhibitor – confirms bypass of ATP synthase)
• Greater effects in mitochondria-rich cells versus mitochondria-poor cells
• Comparison with structurally similar but inactive analogs (if available)
• No effects in cells depleted of mitochondria (ρ0 cells)

Compliance and Safety Information

Regulatory Status:
BAM-SLU MELT Blend is provided as a research chemical for in-vitro laboratory studies and preclinical research only. These compounds have not been approved by FDA, EMA, or other regulatory agencies for human therapeutic use, dietary supplementation, or medical applications. Research use must comply with applicable regulations.

Intended Use:

• In-vitro cell culture studies of mitochondrial function and metabolism
• In-vivo preclinical research in approved animal models with appropriate IACUC protocols
• Laboratory investigation of metabolic regulation mechanisms
• Academic and institutional research applications
• Pharmaceutical research studying mitochondrial uncoupling as therapeutic approach

NOT Intended For:

• Human consumption or administration
• Therapeutic treatment or diagnosis
• Dietary supplementation or weight loss products
• Athletic performance enhancement
• Veterinary therapeutic applications without appropriate regulatory oversight
• Any use outside approved research settings

Safety Protocols:
Researchers must follow standard laboratory safety practices:

• Use appropriate personal protective equipment at all times
• Handle in well-ventilated areas or chemical fume hood
• Follow institutional biosafety and chemical safety guidelines
• Avoid skin contact, inhalation, and ingestion
• Dispose of waste according to hazardous chemical regulations
• Consult safety data sheets (SDS) for specific safety information
• Report exposures to institutional safety officer
• Maintain spill cleanup materials in laboratory
• Post appropriate hazard warnings in work areas

Research Safety Considerations:

• Mitochondrial uncouplers are pharmacologically active compounds that can show significant toxicity at excessive concentrations
• Classical uncouplers (DNP) have caused human fatalities through hyperthermia and metabolic crisis
• While BAM-15 and SLU-PP-332 demonstrate improved safety profiles in research models, treat with appropriate caution as bioactive compounds
• Establish safe concentration ranges in each experimental system before large-scale studies
• Monitor cellular and animal health parameters carefully throughout studies
• Have appropriate emergency response plans for animal studies
• Never taste or smell research compounds
• Store separately from food and beverages

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