Spheroid preparation and immunofluorescence protocol

This protocol provides a guide for preparing and staining 3D spheroids using immunofluorescence techniques. The protocol is also applicable to disease models and tumor spheroids, enabling researchers to study both healthy and pathological conditions in 3D culture systems. Designed to mimic in vivo tissue architecture more accurately than 2D cultures, spheroids offer enhanced biological relevance for drug screening, cancer research, and tissue engineering. The protocol covers spheroid formation, fixation, permeabilization, blocking, antibody staining, and imaging. It includes recommendations for materials, incubation conditions, and troubleshooting tips to ensure reproducibility and high-quality results. Proper sample preparation is emphasized as a critical step for achieving high-quality imaging and reproducible results across different microscopy techniques. Whether using Matrigel or synthetic scaffolds, this guide helps researchers optimize spheroid handling and visualization for various biological assays.

As each spheroid/organoid differs, you will need to optimize the protocol for your specific model; however, this protocol can serve as a starting reference.

Introduction

Spheroid models are increasingly used in biomedical research due to their ability to replicate complex cell-cell and cell-matrix interactions. This protocol enables researchers to generate and analyze spheroids using immunofluorescence, a powerful technique for visualizing protein expression and localization. The protocol also supports functional imaging approaches, allowing for deeper biological insights by linking dynamic cellular processes with static functional markers. The stepwise approach ensures compatibility with various cell types and experimental goals. Fluorescence microscopes are essential for visualizing protein expression and localization in spheroids, providing high specificity and spatial resolution. By integrating scaffold-based or scaffold-free methods, this protocol supports flexible experimental design and high-resolution imaging. It is ideal for scientists seeking to enhance the physiological relevance of their in vitro studies.

Background and principles

Spheroids are 3D aggregates of cells that better simulate tissue microenvironments compared to traditional 2D cultures. Their formation relies on cell self-assembly in non-adherent conditions or specialized platforms like hanging drops or microfluidics. Immunofluorescence staining of spheroids involves fixation to preserve morphology, permeabilization to allow antibody access, and blocking to reduce non-specific binding. This protocol emphasizes the importance of optimizing each step—especially fixation and antigen retrieval—to maintain structural integrity and ensure accurate staining.

Selecting the appropriate excitation wavelength is crucial for achieving an optimal fluorescence signal and minimizing spectral overlap during imaging. Advanced image processing techniques are essential for enhancing image quality and extracting quantitative data from spheroid images.

Spheroids can be prepared from a wide range of cells, such as tumors, embryonic cells, neural tissues, hepatocytes, mammary gland cells, etc., using different methods (non-adherent plates, hanging drop, microfluidics, spinners, agarose molds, bioprinting, etc.). They are increasingly used in drug screening, cancer research, toxicology and tissue bioengineering.

Stage 1 - Spheroid preparation in a 3D environment

Spheroids are 3D in vitro aggregates created using a wide range of methods. The type of 3D culture method will depend on the origin of the tissue/cells, cost, and purpose of the study. Independent of their formation method, spheroids can be included in different extracellular scaffolds to provide an environment that better mimics native tissue architecture. Spheroids can also be analyzed scaffold-free.

Materials required

• Non-adherent tissue culture plates (96-well plates or other)
• Sterile PBS 1% Bovine Serum Albumin (BSA)
• Imaging grade plates (96-well plates or other)
• 3D scaffold

Steps

A wide range of methods can be used to create spheroids. For the round-bottom non-adherent tissue culture plate method, seed cells in wells.

Incubate at 37ºC in a cell culture incubator and verify good spheroid formation visually.

Recover the spheroids using wide-bore ice-cold tips (or cut tips with clean scissors) and place them into ice-cold tubes.

• Tubes should be precoated overnight with sterile 1% Bovine Serum Albumin (BSA)/phosphate-buffered saline (PBS) to reduce cell adhesion to the tubes.

Spin the tubes for 20 seconds at 4ºC (20 x g).

Discard the medium carefully by aspiration.

Add Matrigel™ or another 3D scaffolding material (synthetic scaffolds, collagen, hydrogels etc) to each tube.

Mix very carefully by gently pipetting up and down two times using wide bore tips.

Dispense the scaffold and spheroid mix into the wells of a PDL-coated imaging-grade plate (eg 96-well format).

Incubate for 15 min at 37ºC in a cell culture incubator.

Add warm culture medium (37ºC).

Incubate for 90 min at 37ºC in a cell culture incubator.

Stage 2 - Spheroid fixation

Like in 2D immunocytochemistry, fixation is an essential step in 3D immunofluorescence. The right fixation method preserves and stabilizes the cell morphology and architecture of the spheroid, allowing for the best results. Spheroid fixation deactivates proteolytic enzymes that could degrade the sample and protects the sample against decomposition from microorganisms.

Materials required

• PBS (ab285410)
• 4% paraformaldehyde and/or 100% methanol (chilled at -20°C)

Steps

Remove the culture medium and wash the samples three times using PBS.

Remove PBS and add cell fixative (preferably under a chemical fume hood). Among others, the two following methods could be used:

• 4% paraformaldehyde in PBS pH 7.4 for 10 min at room temperature
• 100% methanol (chilled at -20°C) at 4°C for 5 min

Remove the fixative and wash plates three times with PBS.

Stage 3 - Permeabilization

Incubating the spheroids with a detergent allows antibodies to penetrate cell membranes, which is necessary for immunostaining intracellular targets. Different detergents have different permeabilization properties, some more efficient than others for a particular protein of interest. For example, proteins localized in the mitochondria and/or the nucleus often require the use of a detergent such as Triton X-100 for their detection.

For 3D biological samples in the extracellular matrix, the detergent Triton X-100 is recommended. The detergent used, the optimal percentage and the incubation time will depend on the protein of interest and should be optimized. Methanol fixation will also permeabilize the cells and higher antibody penetration may be achieved using this fixation method.

Materials required

• Permeabilization buffer: PBS 0.5% Triton X-100, PBS 2% Triton X-100, or PBS 10% Triton X-100

Steps

Remove PBS and add permeabilization buffer for one hour at room temperature using a flat shaker.

Stage 4 - Blocking, immunostaining, and imaging

A blocking step is necessary to reduce non-specific antibody binding. Serum and/or an abundant protein buffer is used to saturate potential non-specific binding sites. Ideally, the serotype included in the blocking buffer should match the species of the secondary antibody to achieve the best results. For spheroid immunostaining, all blocking, washing, and immunostaining steps should be performed with constant gentle mixing, eg, using a flat shaker at room temperature.

Materials required

• PBS containing 0.1% Tween, 1% Bovine Serum Albumin, 22.52 mg/mL Glycine, and 10% Goat Serum ab7481
• PBS
• Wash buffer: PBS containing 0.1% Tween
• Primary antibodies
• Secondary antibodies
• PBS containing 0.1% sodium azide

Steps

Remove the permeabilization buffer and add PBS containing 0.1% Tween, 1% Bovine Serum Albumin, 22.52 mg/mL Glycine, and 10% Goat Serum.

• Incubate overnight at room temperature on a flat shaker.

Wash the samples with PBS containing 0.1% Tween (wash buffer).

Add primary antibodies (unconjugated or conjugated) at the desired concentration.

• Titration of the primary/conjugated antibodies may be required to achieve satisfactory immunostaining. Incubate according to the manufacturer’s protocol.

Wash the samples four times with Wash buffer (four washes of one hour each).

If required, add secondary antibodies and/or a nuclear stain (eg, DAPI or Hoechst). Incubate overnight according to the manufacturer’s protocol.

Wash the samples four times with Wash buffer (four washes of one hour each).

PBS and add mounting media or storage buffer (PBS containing 0.1% sodium azide).

Store in the dark at 4ºC until required for imaging.

Image the samples using the appropriate excitation/emission filter sets and/or lasers.

Comparison to other methods

Compared to 2D immunocytochemistry, 3D spheroid immunofluorescence offers superior modeling of tissue architecture and cellular behavior. While 2D cultures are easier to handle, they lack the spatial complexity of spheroids. Organoids provide even more complexity but are harder to standardize. Spheroids strike a balance between physiological relevance and experimental control. This protocol also contrasts scaffold-based and scaffold-free approaches, allowing researchers to choose based on assay requirements. The protocol enables comparison of specific cell populations to other cells within the spheroid, facilitating detailed analysis of cellular features and interactions. The inclusion of antigen retrieval and optimized permeabilization distinguishes this method from simpler staining protocols, improving signal clarity and reproducibility. Molecular genetics approaches can also be integrated for deeper analysis of gene function in spheroid models.

Applications in drug screening

This protocol is applicable across a wide range of research areas, including oncology, toxicology, regenerative medicine, and drug development. The protocol enables the study of cell cycle dynamics and cell division within spheroids. Spheroids derived from tumor cells, hepatocytes, neural tissues, or mammary glands can be used to study invasion, proliferation, and response to treatment. Using this approach, cell cycle arrest and regenerative processes can also be investigated. Immunofluorescence enables precise localization of biomarkers, making it ideal for evaluating therapeutic targets or cellular responses. The protocol supports high-content imaging and quantitative analysis, facilitating translational research and personalized medicine approaches. It is also suitable for validating antibody specificity in 3D contexts.

Limitations of 3D cell cultures

Despite its advantages, spheroid immunofluorescence presents challenges. Antibody penetration can be inconsistent, especially in larger spheroids or dense scaffolds. Fixation may mask epitopes, requiring careful optimization of antigen retrieval. Imaging depth is limited by light scattering and autofluorescence, which may affect signal quality. Additionally, spheroid formation is cell-type dependent and may require empirical adjustment of seeding density and incubation time. The protocol demands meticulous handling to avoid spheroid disruption, and results may vary based on scaffold composition and imaging system used.

Troubleshooting

Common issues include poor spheroid formation, weak staining, and high background. To improve spheroid integrity, optimize cell density and incubation time. If staining is weak, adjust antibody concentration or permeabilization conditions. Background noise can be reduced by refining blocking buffers and wash steps. Ensure fixatives are fresh and used under appropriate conditions. For inconsistent imaging, verify mounting media compatibility and use appropriate filter sets. If epitope masking occurs, incorporate antigen retrieval steps. Always validate antibodies in 3D contexts and perform pilot experiments to refine conditions.