Important Notes for coating surfaces with Laminin
- Ensure the cells are healthy and in adequate number.
- Laminin needs to thaw slowly at 2 - 8 °C. If the product has been warmed up at room temperature and it gels, it cannot be reactivated for use.
- Thawed, unused product may be stored in working aliquots at -20 °C
- Surface coverage concentration may differ with the cells being cultured. Protocol optimization is recommended to determine actual 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 the steps in the protocols need to be performed in sterile conditions.
Laminin coating protocol for culture ware
- Thaw laminin at 2 - 8 °C; avoid rapid thawing to prevent formation of a gel.
- 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.