1. How can the laminin coating be carried out easily and cost-effectively?
Dilute the thawed laminin stock solution with 1xDPBS (Ca++/Mg++) and add the diluted laminin solution to tissue culture-treated cultureware. If this is the first time you use laminins for your cell culture, we recommend to start with a slightly higher coating concentration (see Instruction BL001). The optimal coating concentration is cell type-dependent and should be optimized empirically for specific isoforms and cell lines. Often a lower concentration than the recommended starting concentration could be used. If not used immediately, coated plates and diluted coating solution can be kept at +2°C to +8°C for up to 4 weeks. To prevent evaporation and contamination seal the cultureware (e.g. with Parafilm®). After a few weeks extra 1xDPBS (Ca++/Mg++) might have to be added to prevent the plate from drying out. The laminin matrix will be inactivated if let dry.
2. What is the purity of Laminin products?
The purity of our laminin products is greater than 95% (as assessed by SDS-PAGE).
3. Can I mix the Laminins with other matrix proteins, such as collagen?
We don't have any data for this, but to ensure the best coating, one should do titration to optimize the mix ratio and coating time.
4. How should I store the laminin protein properly?
The laminin stock solution is stable for 2 years when stored at -20°C. Thawed laminin stock is stable for at least 3 months when stored at +2°C to +8°C under aseptic conditions. If desired, the laminin stock can be dispensed into working aliquots and stored at -20°C. Do not re-freeze aliquots after thawing. Laminin coated plates are recommended to be used as fresh as possible but, for your convenience, coated plates and diluted coating solution can be kept at 2-8°C for up to 4 weeks. To prevent evaporation and contamination add extra 1xDPBS (Ca++/Mg++) and seal the cultureware (e.g. with Parafilm®). The laminin matrix will be inactivated if let dry. 521-To-Go™ pre-coated plates (24- and 96-well) with can be stored at room temperature (+15°C to +25°C) with maintained functionality for at least 1 year.
5. According to the BioLamina instructions you can keep the coated plates either in coating solution or DPBS (Ca++/Mg++) at 4°C, which is best or preferred?
Coated plates can be kept in the coating solution. To prevent evaporation and contamination seal the cultureware (e.g. with Parafilm®). If needed, add extra 1xDPBS (Ca++/Mg++) to prevent the coating to dry out.
6. How long can I store the coated plates?
Laminin coated plates are recommended to be used as fresh as possible but, for your convenience, coated plates and diluted coating solution can be kept at 2-8°C for up to 4 weeks. Seal the cultureware (e.g. with Parafilm®) to prevent evaporation and contamination. The laminin matrix will be inactivated if let dry. Adjust the volume by adding DPBS (Ca++/Mg++).
7. How long can you store coated plates at 37°C, i.e. can unused wells still be used after a few days in 37°C?
Unused, coated plates stored at 37°C are not recommended to be re-used.
8. Can I store the thawed laminin protein in 4°C and for how long?
Thawed Laminin stock is stable for at least 3 months when stored at +2°C to +8°C under aseptic conditions. Coated plates and diluted coating solution can be kept at +2°C to +8°C for up to 4 weeks.
9. How stable is recombinant laminin at room temperature?
Laminins are usually quite stable at room temperature (+15°C to +25°C) with proper handling, but one should always try to avoid unnecessary exposure of the protein to ambient temperatures. If possible, keep the sample on ice during work.
10. Can I re-use the coating solution and for how many times?
Due to contamination risks and uncontrollable coating concentration issues we do not recommend to re-use the coating solution but rather to evaluate the optimal coating concentration for you cells and application by titration. Often a lower concentration than the recommended starting concentration could be used. The optimal coating concentration is cell type-dependent and should be optimized empirically for specific isoforms and cell lines.
11. How does the coating concentration affect the properties of the cells?
Too little coating could result in slow growth. No significant impact on cell properties has been observed.
12. How long does the laminin coating last in culture? Do I have to add extra Laminin to long-term cultures?
The laminin coating is functional for at least one month in culture.
13. Is there a difference in performance between the slow coating and the fast coating?
Overnight coating at +2°C to +8°C is recommended since it provides a more reliable coating, however, if a more rapid coating is required a faster coating at +37°C for 2 hours works for most cell lines and applications.
14. Have you noticed the proteins crashing out of solution after repetitive freeze thaw cycles? How many freeze thaws can be done before the complex destabilizes?
Laminins are large trimetric glycoproteins and do not handle repeated freezing and thawing. Thawed laminin stock and is stable for at least 3 months when stored at +2° to +8°C under aseptic conditions. If desired, the laminin stock can be dispensed into working aliquots and stored at -20°C. Do not refreeze aliquots after thawing.
15. Can the laminin be used to coat other surfaces such as glass?
Yes, the LN521™ can be used for coating glass with good cell attachment and maintained function as a result. Coat glassware as you normally coat your cultureware, however, overnight coating at +2°C to +8°C is recommended for a more reliable coating. Seal the coated glassware to avoid evaporation. Make sure all the surface is completely covered by the laminin coating solution as uncoated surface will not support cell growth. Please see Miyanari et al., 2013 for coating reference.
16. Can cells, which have been growing on feeders or Matrigel, be directly transferred onto Laminin? Will cells look the same after transfer?
When moving you cells to LN521 from another feeder-free matrix (i.e. Matrigel), generally no specific adaptation is needed. Passage your cells as a single cell suspension, as described in Instruction BL003, and seed onto LN521 coated plates at a higher cell density (50,000–100,000 cells/cm2) for the first passage. When using LN521 no treatment with apoptosis inhibitors, such as Rho-kinase (ROCK) inhibitor or blebbistatin, is needed. Cell morphology should not change much, however, you should be prepared for a different growth pattern. LN521 promotes high migration which is vital for cell survival. That will render a more even spread of the cells as compared to feeders and other feeder-free matrices. Also the cells will likely grow faster.
When changing from feeders to LN521 some more adaptation will likely be required, see Instrictions for use BL004 for assistance. Cells might display a different morphology for the first one or two passages, and likely due to the packed monolayer the cells form when confluent, rather than thick colonies as seen on feeders. It is important that the cells are of high quality when being transferred from feeders to LN521. LN521 support maintenance of pluripotent cells but may also support some differentiated cells. Therefore it is important that only undifferentiated cell colonies are being transferred.
17. Are your coating protocol and laminin functions optimized for any specific plastic brands?
The LN521 coating is not optimized for certain plastic brands or well formats, however out of the brands tested there are a few with which the laminin coating seem to be functioning better (e.g. Falcon, Sarstedt, Corning) than others. We do not recommend using Nunc plates when coating with laminin.