Laminin is an extracellular matrix multi-domain trimeric glycoprotein, and is the main non-collagenous component of basal lamina that supports adhesion, proliferation and differentiation. Laminin is composed of both A, B1 and B2 chains, which are connected by many disulfide bonds. Laminin supports growth and differentiation of many cell types including epithelial, endothelial, neural, muscle and liver cells. The optimal concentration for cell culture depends on cell type and specific application.
Important Notes for Coating Surfaces with Laminin
- Ensure the cells are healthy and in adequate number.
- Laminin must be thawed slowly at 2 - 8 °C. If the product warms to room temperature and gels, it cannot be reactivated for use.
- Thawed, unused product may be stored in working volumes at -20 °C
- Surface coverage concentration may differ with the cells being cultured. Protocol optimization is recommended to determine optimal coating concentration for your cells. Recommended concentrations are listed in Table 1.
- Do not over-dry culture plates/cover slips post coating as this disrupts the structure of laminin and affects the attachment of cells.
- All steps in the protocols must be performed in sterile conditions.
Laminin Coating Protocol for Cell Culture
- Thaw laminin at 2 - 8 °C; avoid rapid thawing to prevent gel formation.
- Dilute in balanced salt solution to the desired concentration. Solution concentration is dependent upon the application. A typical stock concentration is 0.01%.
- Coat culture surface with a minimal volume.
- Once the entire area is covered, remove excess solution. This can be used to coat another well/plate.
- Air dry at least for 45 minutes before introducing cells and medium.
Laminin Coating Protocol for Neurospheres
- Precoat the coverslips with poly-L-lysine (Product No. P5899) or poly-D-lysine (Product No. P7280) at 100 μg/mL.
- This is incubated for 30 minutes or more at room temperature. The coverslips are washed with sterile water and allowed to dry.
- They can be stored at room temperature until ready to use (up to 1 month).
Coverslips are then coated with 30 - 50 μL of laminin (50 μg/mL) which has been warmed.
- Incubate overnight in a humidified 37 °C, 5% CO2 incubator or at 37 °C for 30 minutes.
- Remove excess laminin and rinse with PBS twice. Alternatively, wash with DME (50 μL) twice.
- Add the cells to the coverslips/slides and culture as needed.
Alternatively, a ready-to-use solution of poly-D-lysine and laminin is available for use with neural stem cells.