CellLight™ Nucleus-GFP, BacMam 2.0 - FAQs

View additional product information for CellLight™ Nucleus-GFP, BacMam 2.0 - FAQs (C10602)

12 product FAQs found

我采用CellLight 标记试剂处理神经元时，转导效率很低，该如何提高转染效率？

与许多其它细胞相比，神经元转导更为困难。提高转导效率的主要方法是采用更多数量的病毒颗粒标记细胞。对于原代神经元，在铺盘时转导要比已培养细胞转导效果更好。此外，蛋白的表达在神经元中发生较慢，表达高峰通常发生于2-3天后而非转导后16小时。

我想用核酸染料来追踪我的细胞，比如DAPI或者Hoechst染料。您有什么建议吗？

这是不推荐的。这些染料与DNA和RNA的结合会影响核酸的正常功能，扰乱转录和增殖。诸如CellTracker染料或Qtracker试剂在不严重扰乱细胞正常活动的条件下对其进行追踪。如果您仍需要使用核酸染料进行标记且细胞是哺乳动物和非血液来源的话，CellLight 细胞核试剂可通过瞬时转染进入细胞，在核表达蛋白上表达GFP或RFP长达数天而不影响其功能。

How can I increase the transduction efficiency with the BacMam 2.0 reagents such as the the CellLight and Premo products?

Try varying particle-to-cell ratio (PPC), incubation volume, temperature and, cell density (if adherent cells are transduced). For adherent cells, we recommend a confluence of about 70%. Following the PPC, adjusting the volume is the next best parameter to change to optimize protein expression. If that doesn't work, you can also use the BacMam Enhancer Kit (Cat. No. B10107).

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

Is there any way to preserve the CellLights labeling beyond 5 days?

Cells transduced with the CellLights reagents can be stored frozen for several months after transduction, without loss of expression.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

Are the CellLights products toxic to cells?

If the viral particles are used at the level we recommend, they are very well tolerated by cells.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

For how long will the CellLights products label my cells?

The BacMam 2.0 CellLights typically express for 5 days after transduction.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

What cell types can the CellLights products be used with?

The first generation BacMam reagents were shown to efficiently transduce over 90 cell types, including stable cell lines and primary cells. With BacMam 2.0, it is now possible to also efficiently transduce primary neurons and stem cells.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

What safety precautions need to be taken to work with the BacMam reagent?

Our BacMam constructs do not replicate in mammalian cells, making them Biosafety Level I (the lowest biosafety level). They can be handled with the standard precautions used for any cell-based reagents.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

I want to label the nuclei of live cells and track them over time. Can I use DAPI for this?

We do not recommend doing this. DAPI is considered to be a semi-permeant/impermeant nucleic acid stain. DAPI staining of live cells may be inconsistent. It is best used as a counterstain for fixed samples. Other cell permeable nucleic acid stains, such as Hoechst or the SYTO dyes may affect cellular function.

For mammalian cells, we recommend using the CellLight Nucleus transduction reagents, available in CFP, GFP and RFP. With these reagents, the cells are transduced overnight in a single labeling step and the next day the nuclei will fluoresce. The label may be retained for 3-5 days and should not affect cell function. Cytoplasmic cell tracking dyes such as the CellTracker dyes may also be used.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

Do CellLight BacMam 2.0 reagents work on all cell types?

BacMam delivery technology works with over 90 mammalian cell types. However, cells with hematopoietic origin do not show effective transduction with CellLight BacMam 2.0 reagents.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

I keep getting low transduction efficiency when using CellLight labeling reagents on my neurons. What can I do to improve the efficiency?

Neurons are more difficult to transduce than many other cells. The main way to improve transduction is to label with a higher number of particles per cell. For primary neurons, it can also help to transduce them at the time of plating rather than on established cultures. There can also be a slower onset of expression in neurons and peak expression often occurs on day 2-3 rather than 16 hours after transduction.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

I want to track my cells with a nucleic acid stain, like DAPI or Hoechst dye. Do you recommend this?

This is not recommended. When these stains bind to DNA and RNA, they may affect the normal function of the nucleic acids, disrupting transcription, as well as replication. Other reagents, such as CellTracker dyes or Qtracker reagents are more optimized for tracking without disrupting normal activity. If a nuclear label is still desired, though, and the cells are mammalian and non-hematopoietic, CellLight nuclear reagents can transiently transfect cells to express GFP or RFP on a nuclear-expressing protein for up to several days without affecting function.

Find additional tips, troubleshooting help, and resources within our Cell Tracing and Tracking Support Center.