Cytoskeleton and Focal Adhesion Organization Assay: An Immunofluorescence-based Method to Study Cell Adhesion and Spreading on Substrates

Overview

This video describes the technique of studying the cytoskeleton and focal adhesions in primary human colon cancer cells to understand its variations depending on substrate rigidity. This culture of cancer cells on soft and hard substrates allows various downstream biophysical measurements for cancer prognosis.

Protocol

1. Immunofluorescence Microscopy Assays

1. Take the substrates with cells to be immunostained to the laminar hood and remove the culture media.
2. Rinse the substrates with PBS and fix the cells with 4% paraformaldehyde in PBS for 20 min at RT.
3. Wash the substrates with PBS three times (5 min each time). Consequently, incubate the substrates with 500 µl signal enhancer for 30 min and rinse with PBS.
4. Incubate cells with the monoclonal anti-vinculin antibody at a 1:250 dilution in PBS for 45 min at RT. Then, wash the substrates with PBS 3 times (5 min each time).
5. Incubate the samples with secondary antibody Alexa Fluor 488 goat anti-mouse IgG at a 1:200 dilution in PBS at RT for 30 min. Wash the substrates with PBS 3 times (5 min each time).
6. To visualize the F-actin structure, incubate cells with TRITC phalloidin conjugates at a concentration of 50 µg/ml for 45 min at RT. Then, wash the substrates with PBS 3 times (5 min each time).
7. Image the samples using a confocal scanning laser microscope (Figure 1A-1D).

Representative Results

Figure 1. Immunofluorescent staining of F-actin and vinculin on soft gel and hard polystyrene substrates. No actin fiber was present in the less spread (A1) tumor cell or (A2) normal cell on soft 2 kPa gels. However, punctate vinculin-containing focal adhesions are present on soft gels (B1-B2). Conversely, primary cells show well-spread morphology, well-defined actin stress fibers (C1-C2), and discrete elongated focal adhesions (D1-D2) on rigid polystyrene substrates.

Materials

Name	Company	Catalog Number	Comments
Reagents
PBS	Lonza	17-516F
Paraformaldehyde	Electron Microscopy Sciences	RT15710
Signal enhancer	Life technologies	I36933
Monoclonal anti vinculin antibody	Sigma-Aldrich	V9131
Alexa Fluor 488 goat anti-mouse IgG	Life technologies	A11001
TRITC phalloidin conjugates	Sigma-Aldrich	P1951
Materials
12 mm2 glass cover slips	Corning	2865-12