This question is part of the following Ask The Expert session:
There are many methods that can be used to fluorescently label and image cells in a Matrigel matrix system. Here we cover three topics: labeling cells in Matrigel matrix assays, immunofluorescence analysis for surface markers (such as those used in 3D culture and culture of pluripotent stem cells), and fixing and embedding cells in Matrigel matrix prior to sectioning.
Staining has been done with all types of Matrigel matrix. However, the use of phenol red-free Matrigel matrix will reduce autofluorescence. We also recommend the use of Falcon® culture slides (Corning Cat No. 354180) for in situ analysis such as immunofluorescence studies. The slides are a specially cleaned and triple rinsed glass with an upper polystyrene chamber.
Labeling Cells in Matrigel Matrix Assays
Labeling is easiest if you culture cells on a thin coat of Matrigel matrix or you image cells from a Matrigel matrix assay such as invasion or tube formation. Most fluorescent methods can be used directly as described by manufacturers. In addition to pre-labeling intrinsic dyes such as green fluorescent protein (GFP), Calcien AM and DiI are frequently used, depending on how long the dye is required to remain stable.
Protocol for labeling cells using Corning Calcein AM dye:
Corning Calcein AM dye is generally used at 8 μg/mL in Hanks Balanced Salt Solution (HBSS). HBSS is recommended because the use of culture medium results in autohydrolysis of the label, which results in unacceptably high backgrounds. Remove medium from the plates being careful to avoid disruption of the matrix by gently aspirating the medium using a Pasteur pipet. Wash the plate with HBSS and repeat the wash a second time. Label the cells by adding 8 μg/mL Calcein AM in HBSS and incubate for 30 to 40 minutes at 37°C, 5% CO2. Gently remove the labeling solution and wash twice with HBSS. The plate is now ready for image acquisition using an automated imager or for taking pictures using a fluorescent microscope. NOTE: Once hydrolysis occurs, Calcein AM leaks out of cells resulting in a higher background. Labeled plates can be stored at 4°C for 1 to 2 hours with minimum increase in background.
Links to Corning procedures are:
Immunofluorescence Analysis with Matrigel Matrix
Immunofluorescence analysis preparation methods using Matrigel as a 3D matrix have been widely published and are represented by this method:
Nat Cell Biol. 2001 Sep; 3(9): 785–792. ErbB2, but not ErbB1, reinitiates proliferation and induces luminal repopulation in epithelial acini. Muthuswamy, et al.
Protocol for immunofluorescence analysis:
Using the above cited method, “structures were fixed either in 2% paraformaldehyde at room temperature for 15 min. or in methanol:acetone (50:50) at -20°C for 10 min. Fixed structures were washed three times in PBS:glycine (130 mM NaCl, 7 mM Na2HPO4, 3.5 mM NaH2PO4, 100 mM glycine) for 15 min. each. The washed structures were blocked first in IF buffer (130 mM NaCl, 7 mM Na2HPO4, 3.5 mM NaH2PO4, 7.7 mM NaN3, 0.1% BSA, 0.2% Triton™ X-100, 0.05% TWEEN® 20) plus 10% goat serum (GS) for 1–2 hr. and subsequently with 2° blocking buffer (IF buffer containing 10% GS and 20 μg ml−1 goat anti-mouse F(ab′)2) for 30–45 min. Primary antibodies were diluted in 2° blocking buffer and incubated overnight at 4°C. Unbound primary antibodies were removed by washing three times in IF buffer for 15 min. each. Anti-mouse or anti-rabbit secondary antibodies coupled with Alexa Fluor® dyes (Molecular Probes) were diluted in IF buffer containing 10% GS and incubated for 45–60 min. Unbound secondary antibodies were washed as described above. Finally, the structures were incubated for 15 min. with PBS containing 5 μM TO-PRO®-3 (Molecular Probes) and 0.5 ng ml−1 DAPI (Roche) before being mounted with the anti-fade agent ProLong® (Molecular Probes). Confocal analyses were performed.”
Corning has also published numerous application notes and posters that include data on immunocytochemical detection. Examples include:
- Maintenance of Human iPS Cells in a Feeder-free Culture System
- NutriStem® hPSC XF Medium Supports Long-term Culture of Human Pluripotent Stem Cells on Corning® Matrigel® hESC-qualified Matrix
- Assay Methods Protocol: Human Embryonic Stem Cell Culture
Protocol for immunohistochemical detection of cell surface markers on human iPS cells cultured on Corning Matrigel hESC-qualified matrix:
Remove culture media from the cultureware. Wash the hES cells twice with 2 mL of PBS. Fix the cells with 1 mL of 4% paraformaldehyde for 20 minutes at room temperature. Wash the cells twice with 2 mL of PBS for 5 minutes. Block the cells with 1 mL of 0.1% BSA, 10% normal goat serum* in PBS at room temperature for 45 minutes to 1 hour. NOTE: For Oct-3/4 staining, permeabilize in 0.1% Triton X-100, and block with 1% BSA, 10% normal rabbit serum in PBS at room temperature for 45 minutes. 7.2.5 During the blocking step, prepare the primary antibody working solution with PBS containing 1% BSA and 10% normal goat serum* to a final desired concentration. After blocking, incubate the cells with 1 mL/well of diluted primary antibody working solution overnight at 2°C to 8°C, or 1 hour at room temperature. Wash the cells three times with 2 mL of PBS containing 1% BSA for 5 minutes each. Dilute the secondary antibody (fluorescence-conjugated) in PBS containing 1% BSA. Incubate the cells with secondary antibody at 1 mL per well for 60 minutes at room temperature in the dark. NOTE: If using pre-conjugated antibody, secondary antibody will not be required. Wash the cells three times with 2 mL of PBS containing 1% BSA for 5 minutes each. Cover the cells with 4 mL of PBS and visualize using a fluorescence microscope.
Fixing and Embedding Cultures in Matrigel Matrix
Rijal and Li have recently published a method for histological studies:
Protocol for histology and immunostaining:
“The cell-laden scaffolds from the tissue cultures were washed twice with ice-cold 1X PBS and fixed in 10% neutral buffer formalin solution for 24–48 hours at 4°C. After rinsing with cold 1X PBS, the 3D cultures were embedded into OCT or paraffin following standard protocols and sectioned at a thickness of 10 μm using a cryostat or a microtome. For the sections produced using the paraffin fixation, a deparaffinization and rehydration process was performed, followed by antigen retrieval using the tris-EDTA buffer [10 mM tris base, 1 mM EDTA solution, and 0.05% TWEEN 20 (pH 9.0)]. The sections were washed several times with water, stained with H&E or IF antibodies (corresponding primary and Alexa fluorophore–conjugated secondary antibodies) as described previously (Circ. Res. 100, 79–87 (2007), and imaged using light or fluorescence microscopy for further analysis.”