Tips and Best Practices for Metabolite Detection in 3D Cell Cultures

Promega Corporation

Publication date: October 2025

Introduction

Accurate measurement of phenotype is central to understanding how cells function and respond to their environment. This is particularly true for three-dimensional (3D) models such as spheroids and organoids, which provide greater physiological relevance than traditional monolayer cultures while also introducing additional complexity. Careful monitoring of cell health and metabolism is essential to generate phenotypic readouts that are both meaningful and reproducible.

This guide highlights practical considerations, tips, and case studies for using Promega’s bioluminescent metabolite assays with 3D models, helping researchers design experiments with confidence.

Promega is a leader in 3D assay solutions. Whether it be viability, cytotoxicity, or metabolite detection, Promega offers powerful and sensitive tools for your research needs. For more information about 3D models and their applications, visit our 3D Cell Culture Guide.

Explore the complete list of Promega's 3D compatible assays.

Promega Bioluminescent Metabolite Assays

Promega’s bioluminescent metabolite assays provide sensitive and reliable tools for monitoring key metabolic pathways, including glucose utilization, lipid metabolism, amino acid turnover, and mitochondrial function. These assays are based on a simple principle: metabolite-specific dehydrogenases generate NAD(P)H, which drives conversion of a proluciferin substrate to luciferin. In the presence of luciferase, this produces light directly proportional to the amount of metabolite in the sample (Figure 1).

Figure 1. Metabolite Assay Chemistry. We use a novel proluciferin reductase substrate that, in the presence of reduced dinucleotides NAD(P)H, is converted to luciferin, which is subsequently used by luciferase to produce light. By coupling this core technology with selective metabolite dehydrogenase enzymatic conversion, we built a set of metabolite detection assays where light output is proportionate to the metabolite level in the sample.

For researchers working with 3D models, this format offers distinct advantages:

High sensitivity and broad dynamic range. Detection spans femtomole levels to millimolar concentrations, capturing subtle changes in small sample volumes.
Wide assay window. Light output exceeds background by more than 100-fold, providing confidence in distinguishing true biological effects.
Simple workflow. An add-and-read format minimizes handling, reduces variability, and works seamlessly with standard plate readers (Figure 2).

View a complete list of Promega Metabolic activity assays.

Figure 2. Simple workflow for metabolite detection using Promega assays.

Choosing the Right Media for 3D Metabolite Assays

Culture medium is a central experimental variable in metabolism studies. Beyond supporting cell growth, medium defines the metabolite environment cells encounter and therefore strongly influences both cellular behavior and assay readouts. This influence is amplified in 3D systems, where gradients of oxygen and nutrients form within spheroids or organoids, making medium formulation especially critical. The MISpheroID Consortium has underscored the need for careful reporting of media conditions to ensure transparency and reproducibility in 3D culture research (Nature Methods, 2021). To learn more about the purpose of the MISpheroID knowledgebase, read the following blog.

Nutrient concentrations matter

Commercially available media differ widely in the concentrations of key metabolites such as glucose, glutamine, and pyruvate. For example, glucose levels may range from a physiologically relevant 5 mM to a supraphysiological 25 mM, dramatically altering metabolic behavior of cells.

Serum choice impacts sensitivity

Serum choice adds another layer of complexity: standard fetal bovine serum (FBS) contains millimolar levels of small metabolites such as lactate and glutamate, which can mask subtle cellular changes. Dialyzed serum reduces these background metabolites and improves detection of consumption or secretion events. When feasible, a defined minimal medium supplemented with specific metabolites and dialyzed serum offers the most controlled conditions.

Stay within the assay’s linear range

Equally important is knowing the baseline metabolite concentrations in your medium. Promega metabolite assays provide accurate quantitation in the 50–200 µM range, while many metabolites in common media formulations are present at millimolar levels. Without proper dilution, samples will fall outside the linear detection window and obscure true biological changes. Adjusting dilution factors ensures accurate quantitation and meaningful comparisons across experiments.

Best practices when selecting media for subsequent metabolite assay measurement

Choose physiologically relevant baseline concentrations when possible (e.g., 5 mM glucose).
Use dialyzed serum to minimize background metabolite noise.
Record and report exact media formulations for reproducibility.
Dilute culture supernatants to bring metabolite concentrations into the assay’s linear range.

Practical Considerations for Spheroid Handling

Promega’s add-and-read bioluminescent metabolite assays are well-suited for automated and high-throughput workflows. However, the unique handling challenges of spheroids require some adjustments to ensure reproducible results that scale for high-throughput workflows.

3D Cell Models Sensitive to Media Removal

Changing culture medium in wells containing individual spheroids is technically difficult. In 96-well ultra-low attachment plates, attempts to fully remove medium often disturb or even remove spheroids, leading to well-to-well variability. This is especially problematic for inhibitor or compound screening, where consistency across replicates is critical.

Partial Media Exchange Balances Data Quality with Spheroid Integrity

To address spheroid handling challenges, we suggest a partial exchange approach, removing only 50% of the medium and replacing it with fresh medium containing inhibitors. This strategy reduces the risk of spheroid disturbance and minimizes well-to-well variation, making the workflow more automation-friendly. A limitation of partial medium replacement is that starting metabolite concentrations remain elevated at the time of treatment, reducing the sensitivity for detecting subsequent changes. Despite this reduced sensitivity, the partial exchange method provides a practical balance between data quality and spheroid integrity, particularly in screening contexts.

Best Practices for Medium Exchange in Spheroid Assays

Instead of fully removing culture medium, replace only ~50% of the volume to preserve spheroid integrity while still providing fresh nutrients or drug exposure.
Partial exchange leaves higher baseline metabolite levels, which can reduce sensitivity for detecting small changes.
Media-only and time-zero controls are essential to distinguish treatment effects from baseline metabolite accumulation.

Case Study: Monitoring metabolite consumption and secretion in 3D Models

To illustrate how medium composition and cell density shape assay outcomes, we compared glucose consumption and lactate secretion in HCT116 spheroids grown under two media conditions:

M1: DMEM with 5 mM glucose and 5% dialyzed serum
M2: DMEM with 25 mM glucose and 10% FBS

HCT116 cells were plated at different starting densities (1,000, 2,500, or 5,000 cells per well) in Ultra-Low Attachment plates to enable spheroid formation. After 72 hours, culture supernatants were collected from each well and analyzed using the Glucose-Glo™ Assay and Lactate-Glo™ Assay. Because baseline glucose concentrations in M2 medium were substantially higher than in M1, samples were diluted 800-fold (M2) or 160-fold (M1) with PBS to bring metabolite levels into the linear detection range of the assays.

At higher cell densities (2,500 and 5,000 cells/well), both M1 and M2 media revealed clear shifts in glucose consumption and lactate production compared to controls (Figure 3). At the lowest density (1,000 cells/well), however, changes were detectable only in M1. The lower baseline glucose concentration and reduced background metabolites in M1 created a more sensitive assay window, allowing subtle changes to be measured that were masked in M2.

Figure 3. Glucose consumption and lactate secretion in HCT116 spheroids. HCT116 cells were plated at varying cell densities (1,000, 2,500 and 5,000 cells/well) in 100 ul M1 medium (DMEM with 5 mM glucose and 5% dialyzed serum) or M2 medium (DMEM with 25 mM glucose and 10% FBS) in Corning Ultra Low Attachment (ULA) 96-well plates. A set of wells containing medium only without cells was included as a control. The cells were incubated for 72 hours to allow spheroid formation, then a 20µl sample of culture supernatant was removed from each well and combined with 80µl PBS before freezing the samples for storage. On the day of running the metabolite assays, the samples were thawed and further diluted with PBS (1:40 the original concentration of the culture medium for M1 and 1:200 for M2). Next, 9µl of diluted sample was transferred to selected wells of white opaque low volume 384 well plates and combined with 9µl of Detection Reagent from the Glucose-GloTM Assay (Promega Cat. # J6021) (A, B) or 9µl of Detection Reagent from the Lactate-GloTM Assay (Promega Cat. # J5021) (C, D).

Converting luminescence signals to absolute concentrations highlighted distinct patterns of metabolite use (Figure 4). In M2 medium, glucose declined only modestly (25 mM to ~20 mM in high-density spheroids), and consumption rates remained steady at ~1.3–1.5 nmol/h/cell across densities. In contrast, in M1 medium, glucose was nearly depleted at high density (5 mM to ~2 mM), and consumption rates dropped from ~1.5 nmol/h/cell at low density to ~0.75 nmol/h/cell at high density. Lactate secretion followed the same trend: rates were higher and more consistent in M2 (~1.3 nmol/h/cell) compared to M1 (as low as 0.8 nmol/h/cell at high density). These results show that higher metabolite concentrations, as in M2, can sustain steady metabolic activity in larger spheroids.

Figure 4. Quantification of glucose consumption (A, B) and lactate secretion (C, D) in HCT116 spheroids. Standard curves for glucose and lactate were run in parallel to convert luminescence signals into absolute concentrations. Data represent five biological replicates, with error bars showing standard deviation.

This comparison underscores the dual role of medium composition in shaping assay outcomes. Lower metabolite concentrations, as in M1, enhance sensitivity for detecting subtle metabolic changes, particularly at lower spheroid densities. In contrast, higher metabolite concentrations, as in M2, provide the nutrient supply needed to maintain cell health and consistent metabolic activity in larger spheroids. As a result, both medium composition and cell density must be carefully considered when designing and interpreting metabolic assays in 3D models.

Case Study: Utilizing Metabolite Assays for Inhibitor Screens using 3D Models

Promega’s add-and-read bioluminescent metabolite assays are well-suited for automated and high-throughput workflows. Their sensitivity allows miniaturization to 384- or even 1536-well formats, making them effective tools for high-throughput metabolic screening in complex 3D models. To demonstrate this, we applied the assays in a focused inhibitor study using media collected from spheroids.

We selected two well-characterized metabolic pathway inhibitors to apply to the HCT116 spheroids. The glycolysis inhibitor 2-deoxy-D-glucose (2DG), a nonhydrolyzable glucose analog, was used to block lactate production, measured with the Lactate-Glo™ Assay in 384-well format. The glutaminase inhibitor BPTES, which prevents conversion of glutamine to glutamate, was used to block glutaminolysis, with changes measured by the Glutamate-Glo™ Assay, also in 384-well.

At the start of treatment (“0h”), lactate and glutamate levels in spheroid wells were already higher than media-only controls, reflecting secretion during spheroid formation and the use of a partial (50%) medium exchange strategy. Over 24 hours of treatment, untreated spheroids continued to secrete metabolites: lactate levels rose by ~200% (an increase of ~1.4 mM) and glutamate by ~170% (an increase of ~46 µM) (Figure 5A,B). In contrast, 2DG treatment nearly abolished lactate secretion (>90% inhibition), while BPTES reduced glutamate secretion by ~65–70% (Figure 5C,D).

To confirm that changes in metabolite levels reflected pathway inhibition rather than cytotoxicity, we measured viability using the RealTime-Glo™ MT Cell Viability Assay. Luminescent signals from treated spheroids were comparable to untreated controls, demonstrating that 2DG and BPTES were not toxic under these conditions (Figure 5E).

These results show that Promega’s metabolite assays can be effectively applied to inhibitor testing in 3D spheroids in a miniaturized format, enabling robust detection of pathway-specific effects while maintaining compatibility with automation and high-throughput workflows.

Keys for Success in Measuring Metabolites in 3D Models

Promega’s luminescent metabolite assays provide a sensitive and versatile platform for monitoring metabolic activity in 3D cell models. By coupling metabolite-specific dehydrogenases with NAD(P)H-dependent bioluminescence, these assays deliver femtomole-level sensitivity, a wide linear range, and a robust assay window. To maximize their impact in spheroid and organoid studies, researchers should keep the following factors in mind:

Optimize medium composition. Select physiologically relevant nutrient levels (e.g., low glucose) and use dialyzed serum to minimize background metabolites that mask subtle changes. Aligning culture conditions with experimental goals increases both sensitivity and biological relevance.
Consider cell density effects. Low-density spheroids are more amenable to detecting subtle changes, while higher densities may require nutrient-rich media to maintain steady metabolic activity. Pilot experiments at multiple densities can help identify optimal conditions.
Use partial medium exchange for handling. Replacing ~50% of the medium preserves spheroid integrity and reduces well-to-well variability. While this reduces assay sensitivity, it provides a reproducible and automation-friendly workflow.
Pair metabolite assays with viability measurements. Running assays such as RealTime-Glo™ MT Cell Viability in parallel ensures that observed changes reflect pathway modulation, not cytotoxicity.
Leverage scalability for efficient high-throughput studies. The add-and-read format and luminescent readout make these assays highly compatible with 384- and 1536-well formats, supporting efficient inhibitor screens and other high-throughput applications.

Citations

Peirsman, A., et al (2021). MISpheroID: a knowledgebase and transparency tool for minimum information in spheroid identity. Nature Methods, 18(11), 1294–1303. https://doi.org/10.1038/s41592-021-01291-4

Introduction Promega Bioluminescent Metabolite Assays Choosing the Right Media for 3D Metabolite Assays Practical Considerations for Spheroid Handling Case Study: Monitoring metabolite consumption and secretion in 3D Models Case Study: Utilizing Metabolite Assays for Inhibitor Screens using 3D Models Keys for Success in Measuring Metabolites in 3D Models Citations Related Resources

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