Laminin Coating Protocol for Cell Culture

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

1. Thaw laminin at 2 - 8 °C; avoid rapid thawing to prevent gel formation.
2. Dilute in balanced salt solution to the desired concentration. Solution concentration is dependent upon the application. A typical stock concentration is 0.01%.
3. Coat culture surface with a minimal volume.
4. Once the entire area is covered, remove excess solution. This can be used to coat another well/plate.
5. Air dry at least for 45 minutes before introducing cells and medium.

Laminin Coating Protocol for Neurospheres

1. Precoat the coverslips with poly-L-lysine (Product No. P5899) or poly-D-lysine (Product No. P7280) at 100 μg/mL.
2. This is incubated for 30 minutes or more at room temperature. The coverslips are washed with sterile water and allowed to dry.
3. 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.
4. Incubate overnight in a humidified 37 °C, 5% CO₂ incubator or at 37 °C for 30 minutes.
5. Remove excess laminin and rinse with PBS twice. Alternatively, wash with DME (50 μL) twice.
6. 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.

Table 1. Laminins for Cell Attachment

Product No.	Description	Coating Conc.	Dilute in
L2020	Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane	1-2 μg/cm2	Sterile balanced salt solution or sterile water
L4544	Laminin from human fibroblasts	1-2 μg/cm2	Sterile HBSS or sterile water
11243217001	Laminin from mouse Engelbreth-Holm-Swarm (EHS) sarcoma	2-5 μg/cm2	Basal medium (without serum)
L6274	Laminin from human placenta	1-2 μg/cm2	Sterile balanced salt solution
CC095-M	Laminin, mouse purified	2 μg/cm2	Basal medium (without serum)
CC145	Rat Laminin-5	1 μg/cm2	Basal medium (without serum)
SCR127	Synthetic Laminin Peptide for Rat Neural Stem Cells	13-15 μg/cm2	Sterile water
AG56P	Human Laminin (pepsinized) Purified Protein	1-2 μg/cm2	Sterile PBS
127-2.5	Poly-D-Lysine & Laminin (2.5 mL)	0.25 mL/cm2	Ready-to-use
ECM103	Millicoat Human Laminin Coated Strips (96-Wells)		Ready-to-use

Frequently Asked Questions When Coating Surfaces with Laminin

• What ECM protein is the best for my cells?
  Laminin and fibronectin are widely used extracellular matrix (ECM) proteins; however, for best results, experimentally determine the ideal ECM for each cell line. Stem cells express varied integrins on the surface during different stages of differentiation. The choice of ECM protein depends on the stage of differentiation you are studying.
  
  
• What is the purity of laminin products?
  We do not determine purity of laminin solutions. An SDS-PAGE gel is performed and the pattern (three bands) is comparable to past lots to ensure consistent quality.
  
  
• What is the best solvent to dilute laminin?
  The solvents that are compatible to dilute laminins are provided in Table 1.
  
  
• Can laminin be reused?
  Reuse of laminin is not recommended as there will be changes in the sterility and concentration and structure of laminin. Diluted, unused laminin can be stored at 2 - 8 °C for up to 1 week. Laminin-coated plates can be stored at 2 - 8 °C for up to 4 weeks provided they are sealed well to prevent contamination and/or drying. Do not use the product if discoloration or web-like formations appear on the surface of the coated material.
  
  
• What is the optimum concentration of laminin for coating?
  The coating concentration of our laminins are suggested in Table 1. For best results, optimize the concentrations for your specific cells.
  
  
• What is the best ECM protein to study neural differentiation?
  Laminin or fibronectin along with poly-D-lysine or poly-L-lysine support in vitro neural differentiation well. Refer to the protocol for detailed steps to induce neural differentiation.