Laminin 521: Recommended Protocol

1. Thawing:
   1. Gently thaw Laminin 521 within the temperature range of 2°C to 8°C.
   2. During extended operations, keep the product on ice.
   3. For extended storage, consider dividing the thawed stock solution into smaller aliquots and store them frozen.
   4. Note: Avoid repeated freeze-thaw cycles.
2. Dilution:
   1. Use DPBS (with Ca2+ and Mg2+) to dilute the product to a final concentration between 5-10 μg/ml.
   2. For cells exposed to this product for the first time, consider using higher coating concentrations during initial passages until adaptation.
   3. Note: Determine the optimal Laminin 521 concentration based on cell type and application. For a starting point, we recommend a coating concentration of 0.5 μg/cm^2 on the culture surface. The formula below can guide the dilution process:
      1. Working concentration = Coating concentration x (Culture surface area / Required volume for surface area)
      2. Dilution factor = Stock concentration / Working concentration
      3. The coating concentration and volume may be adjusted based on specific experimental requirements.
3. Handling:
   1. Gently mix the solution. Avoid vortexing.
4. Application:
   1. Promptly apply the diluted Laminin 521 to the cultureware. Suggested volumes for various cultureware are:
      1. 6-well plate: 1 ml/well
      2. 12-well plate: 0.5 ml/well
      3. 24-well plate: 0.2 ml/well
      4. T-25 cm^2 flask: 2.5 ml/flask
      5. T-75 cm^2 flask: 7.5 ml/flask
      6. 35 mm dish: 0.8 ml
      7. 60 mm dish: 2 ml
      8. 100 mm dish: 5.5 ml
   2. Note: The mentioned volumes are based on a 5 μg/ml concentration. Adjust based on the actual bottom area used.
5. Even Spreading:
   1. Gently oscillate the cultureware to distribute the Laminin 521 solution uniformly.
6. Incubation:
   1. Seal the cultureware to avert Laminin 521 solution evaporation.
   2. For an overnight incubation, keep it at 2-8°C. For a quicker coating, incubate at 37°C for at least 2 hours. Prepared cultureware can be pre-coated, sealed, and stored aseptically at 2-8°C for 2-4 weeks.
   3. Note: Prevent the culture surface from drying, as it will deactivate the matrix coating.
7. Final Step:
   1. Remove the Laminin 521 just before cell plating.
   2. Note: Rinsing is not necessary before use.