FAQs
Frequently Asked Questions
Categories
General Information
Does Quick-Tissue™ technology leave a genetic footprint?
Sendai virus (SeV) is an RNA virus, so it does not integrate into the genomic DNA. In principle, a foreign gene introduced intracellularly in the form of RNA is quickly translated and expressed because, unlike DNA, RNA does not need to enter the nucleus for forced expression, thereby providing no chance of mutagenesis. This is discussed in the following review paper: Yamamoto, et al., (2009) "Current prospects for mRNA gene delivery." Eur. J. Pharm Biopharm 71, 484-489.
Will SeV remain active after differentiation?
No. The SeV used in our kits is a temperature-sensitive mutant that is active at 33℃ but becomes inactive at 37℃, which is the temperature instructed in the user guides post-differentiation.
Is Sendai virus (SeV) dangerous?
SeV is not pathogenic to humans (i.e., humans are not the natural host of the virus) and the infection does not persist in immunocompetent animals. Furthermore, SeV used in our kits does not produce infectious viral particles upon transduction to host hPSCs and is a temperature-sensitive mutant, such that it is active at 33℃ but becomes inactive at 37℃. However, because SeV can be transmitted by aerosol and contact with respiratory secretions and is highly contagious, appropriate care must be taken to prevent potential mucosal exposure to the virus. Our SeV-based kits must be used under Biosafety Level 2 (BL-2) containment with a biological safety cabinet or a laminar flow hood and with appropriate personal protective equipment. In the event that the virus comes into contact with skin or eyes, decontaminate the affected area by flushing with plenty of water and follow the safety manual prepared by your laboratory and approved by your Institutional Biosafety Committee.
Do I need a license agreement for any of Elixirgen Scientific’s products?
No. You don't need any licence or material transfer agreement (MTA) to use our differentiation kits or iPSC-derived cells. However, please be advised that these products are for research use only.
User Guide Information/Troubleshooting
What should I do if I find on day 1 that cells were accumulated in the center of the well?
It is not recommended to start mRNA-based differentiation induction because the differentiation efficiency is expected to be quite low. For successful differentiation using our mRNA-based methods including Mesendoderm Booster, hPSCs should be evenly distributed in the well. It is likely that the plate was shaken too much, and cells were accumulated in the center of the well while they were floating. Next time, please handle the plate more gently or it can be left on a flat bench top without vibration for 15 min before it is put inside a CO₂ incubator.
Why does a reagent in my differentiation kit have less than the required volume?
For reagents supplied in small volumes, be sure to briefly centrifuge before use. If pipettors have been calibrated recently and the volume is less than it is supposed to be even after the tube is spun down, please contact us at [email protected].
Why do I see many floating cells and/or cellular debris on day 1?
hPSCs were likely damaged while they were harvested. hPSCs may have been mechanically harvested by scraping or were pipetted too much or too vigorously. Lift Solution D1-treated hPSCs only by pipetting them up and down up to 15 times. The mixing of hPSCs with SeV may have also been done too strongly. Gently rock the plate several times to mix hPSCs with SeV.
How long do I need to maintain hPSCs prior to beginning to use your kit?
Before beginning differentiation, cultures should be healthy and displaying typical morphologies (i.e., round colonies with tightly packed cells exhibiting a small cytoplasmic area), few spontaneously differentiating cells, and normal growth rates (i.e., doubling once a day). We recommend growing the cells for at least 14 days post-thaw before differentiating and not allowing the cultures to reach more than 80% confluency.
