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Maggie Bach and Dan Lazar 

Promega Corporation
Publication Date: December 2019


As researchers increasingly adopt 3D cell culture systems to study disease and cellular model systems, assays originally intended for use with monolayer cell cultures need to be adapted to more complex 3D systems. While the use of these models provides advantages, such as increased physiological relevance to in vivo tissues, they also present challenges for researchers.

A key feature of 3D models that is both an advantage and challenge is heterogeneity within the model. The nutrient and oxygen gradients present in a 3D model are very different from the consistent nutrients and oxygen levels available to cells grown in monolayer. These gradients impact cell growth in a 3D model, causing cells in the center of a 3D model to be necrotic or quiescent, while cells on the outer layer of the spheroid continue to proliferate. Each cell model will be different depending on the size, cell types and technique used to form the structure. This heterogeneity helps researchers better model in vivo tissues. However, the different cell populations and 3D structures present challenges when analyzing how cells in a 3D model respond to treatment.

Case #1: ATP Viability Assay—CellTiter-Glo® 3D

Assay Overview

The CellTiter-Glo® Cell Viability Assay detects live cells by quantitating the amount of ATP present in living cells. The reagent quickly lyses the cells, stabilizes the ATP present, and generates a luminescent signal proportional to the amount of ATP present. The signal is directly proportional to the number of living cells in the sample.


3D structures are much larger with additional extracellular matrix proteins compared to cells grown in monolayer culture. Does CellTiter-Glo® reagent completely lyse the 3D structures and measure all the ATP present in the 3D structures?

Adaptation for 3D models

  1. Reformulated reagent. Promega scientists adapted the reagent formulation by increasing its lytic capacity, and a new CellTiter-Glo® 3D is now available for researchers to use specifically with 3D models. Increased detergent in the reagent ensures that it can lyse 3D structures up to 500μM. The UltraGlo™ Luciferase in the reagent is highly stable compared to native firefly luciferase, and can withstand the increased detergent formulation without a significant impact on activity or stability.
  2. Protocol updates. The new protocol for CellTiter-Glo® 3D includes increased shaking time compared to the original CellTiter-Glo® Assay. This increased shaking time helps physically disrupt the 3D structures.


Research and development scientists extensively verified performance of the reformulated reagent, along with the optimized protocol using multiple 3D models and orthogonal techniques to measure cell number and ATP recovery. We tested HCT116 colon cancer spheroids formed using the hanging drop method and additional cell types, including HEK293 embryonic kidney cells and HepG2 liver carcinoma cells. Orthogonal techniques to answer the question of complete cell lysis included measuring cell death after reagent addition and extracting ATP with acid.

R&D scientists first looked under the microscope at the 3D structures after adding CellTiter-Glo® 3D (a good habit for cell biology researchers). From the images, it looked like the 3D structures were still present even after adding the reagent and additional shaking. This might surprise scientists used to looking at lysed monolayer cells. However, the seemingly intact structure simply means that the extracellular matrix present around the cells was not broken up by the lytic reagent—not that cell lysis was ineffective. Therefore, verification of cell lysis would have to be done with a technique other than simple visual observation of the structure.

Case #2: Reporter Assay—Autophagy LC3 HiBiT Reporter

Assay Overview

Reporter assays introduce a reporter gene into cells and detect the product of the reporter gene when the gene is transcribed and translated by the cell. The reporter can include a protein tag or an enzyme such as luciferase. In this example, the LC3 protein, widely used as a marker of autophagic activity, is tagged with HiBiT, a small 11a.a. peptide tag. Cells expressing the Autophagy LC3 HiBiT Reporter are treated with inducers or inhibitors of autophagy. At the end of the experiment, cells are lysed and total LC3 is quantitated by adding the NanoGlo® HiBiT Lytic Detection Reagent, which includes the complementing LgBiT protein and NanoLuc® substrate.


The Autophagy LC3 HiBiT Reporter was introduced into HEK293 cells, and the reporter cells were grown in both monolayer and 3D spheroids. Does the LC3 HiBiT Reporter signal accurately reflect reporter levels in 3D spheroids?

Adaptation for 3D Models

In this assay, to detect and quantify the tagged protein, the cells must be fully lysed to recover the tagged protein from all the cells. In order to facilitate lysis of the 3D spheroids, the protocol was adapted to increase the shaking time and reagent processing time after addition of the lytic reagent. The 2-minute shaking plus 10-minute incubation for monolayers was increased to 30 minutes of spheroid shaking and incubation.


The performance of the Autophagy LC3 HiBiT Reporter Assay System in 3D models was verified by comparing the recovery of the reporter to the ATP content in the spheroid model, wherein ATP is a useful surrogate for cell number. Different size spheroids were created from the HEK293 reporter cells by seeding increasing cell number in Corning® Ultra-Low Attachment 96-well plates. After 4 days of culture, spheroids of a wide range of sizes were generated for subsequent assay of autophagy reporter levels with the NanoGlo® HiBiT Lytic Detection Reagent. In parallel plates, a similar set of spheroids was processed with CellTiter-Glo® 3D to determine ATP content.


These two cases demonstrate two approaches for adapting assays for use with 3D models. With the increase of 3D model use and high demand for assays formulated for 3D models, more assays will be designed specifically to meet the needs of 3D cell cultures. When that isn’t possible, protocols for existing assays should be modified, and results should be compared to an assay extensively verified for use with multiple 3D models.

Have questions about verifying an assay with your 3D model?