Laminin Coating Protocol

Important points to remember before start

  1. Ensure the cells are healthy and in adequate number.
  2. 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.
  3. Thawed, unused product may be stored in working aliquots at -20°C
  4. 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.
  5. Do not over-dry culture plates/cover slips post coating as this disrupts the structure of laminin and affects the attachment of cells.
  6. All the steps in the protocols need to be performed in sterile conditions.

Laminin coating protocol for culture ware

  1. Thaw laminin at 2 - 8 °C; avoid rapid thawing to prevent formation of a gel.
  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.

Click for ready-to-use, Laminin-coated, 96-well strips.

Laminin coating protocol for neurospheres

  1. Precoat the coverslips with poly-L-lysine (P5899) or poly-D-lysine (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 & Laminin is available for use with neural stem cells.
 

Product # 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 ug/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

Table 1: Laminins for cell attachment

Click for Laminin Mimetics

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Frequently Asked Questions (FAQs)

  1. What ECM protein is the best for my cells?
    While laminin and fibronectin are widely used ECM proteins, the one that your cell needs should be determined by test experiments. Stem cells express varied integrins on the surface during different stages of differentiation. The choice of ECM protein would depend on the stage of differentiation you are interested in studying.

  2. What is the purity of laminin products?
    We do not determine purity of laminin solutions. An SDS PAGE gel is run and the pattern (3 bands) is comparable to past lots to ensure consistent quality.

  3. What is the best solvent to dilute laminin?
    The solvents that are compatible to dilute laminins are provided in Table 1

  4. 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 kept 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 up of laminin. Do not use the product if discoloration or spider web formations appear on the surface of the coated material.

  5. What is the optimum concentration of laminin for coating?
    The coating concentration of our laminins are suggested in Table 1. However, optimizing the concentrations that work for your cells is recommended.

  6. 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.

    For additional questions contact our technical services here.