Cytoskeleton and Focal Adhesion Organization Assay: An Immunofluorescence-based Method to Study Cell Adhesion and Spreading on Substrates
Abstract
Source: Ali, M. Y et al., Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry. J. Vis. Exp. (2015)
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
- Take the substrates with cells to be immunostained to the laminar hood and remove the culture media.
- Rinse the substrates with PBS and fix the cells with 4% paraformaldehyde in PBS for 20 min at RT.
- 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.
- 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).
- 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).
- 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).
- 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.
Divulgazioni
The authors have nothing to disclose.
Materials
| 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 |
