Immunofluorescent Staining of Spheroids: A Technique to Analyze Spheroids for Expression of Intra- and Extracellular Antigen Expression

- Spheroids are three-dimensional in vitro cell culture models. For immunofluorescent staining, first, generate a spheroid culture of the required cell type in a multiwell plate. Using a micropipette with a wide orifice tip, collect the spheroids, taking care to avoid rupturing due to shear forces.

Next, fix and permeabilize the spheroids to render individual cells permeable. Now, incubate with a solution containing proteins to block non-specific protein interaction sites on the cell surface. Treat the spheroids with a desired primary antibody cocktail. Incubate for the antibodies to bind to the target molecules on the cell surface.

Next, label the spheroids with a fluorescently labeled secondary antibody solution specific to the primary antibodies. Finally, treat the spheroids with a suitable fluorescent DNA-binding dye to stain cell nuclei. Using a laser-scanning fluorescence microscope, image multiple optical cross-sections of the spheroid at different optical planes.

The captured images reflect fluorescently-labeled nuclei and antigens of interest. Merge the stacks using relevant software to obtain the final composite image of the 3D spheroid. In the following protocol, we will perform immunofluorescent staining of spheroids of pancreatic fibroblasts and cancer-associated fibroblasts cultured in the presence of a stimulant, TGF beta 1.

- First, cut off the end of a pipette tip to enlarge the orifice. After the incubation is complete, use this cut pipette tip to collect the spheroids into one 1.5 milliliter reaction tube, per experimental condition or per protein to be analyzed. Centrifuge the spheroids at 1,000 times g for about 30 to 60 seconds. Carefully remove the methylcellulose containing supernatant by pipetting, making sure to not disturb the pelleted spheroids.

- Special care is to be taken when transferring the spheroids to the reaction tubes. Make sure you don't have shear stress forces by cutting the tip. It's also important that you collect all the spheroids. During the washing step, make sure you don't lose the spheroids by aspiration.

- To wash the spheroids at 50 microliters of 1X PBS, centrifuge at 1,000 times g for 30 to 60 seconds, then carefully remove the PBS by pipetting. Depending on the protein to be stained, fix the spheroids either with 50 microliters of 4% paraformaldehyde in PBS for 20 minutes at room temperature.

Wash the spheroids with PBS as previously described. Permeabilize the cells with 0.1% Triton X in PBS for 4 minutes at room temperature. Then wash the spheroids in PBS three times, as previously described. Add PBS containing 5% FBS and 2% BSA to the spheroids and incubate at room temperature for one hour to block non-specific protein interaction sites.

Now, incubate the spheroids with 30 microliters of primary antibodies in PBS with 2.5% FBS and 1% BSA at 4 degrees Celsius overnight. The next day, wash the spheroids in PBS three times as described previously.

After this, incubate the spheroids with 30 microliters of secondary antibodies in PBS with 2.5% FBS and 1% BSA at room temperature for 90 minutes. Wash the spheroids in PBS three times as previously described.

Next, incubate the cells with 30 microliters of DAPI staining solution for 4 minutes at room temperature. Wash the spheroids in PBS one time as previously described. Using a glass Pasteur pipette, place the spheroids on a glass slide in a drop of PBS. Use a laser scanning microscope to image the spheroids by fluorescence microscopy at a magnification of 10 times. Take different optical cross-section of the spheroid at different optical planes of a Z-stack.