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Why do you recommend 7AAD instead of PI staining for cell determination in mouse macrophages?

The results obtained using 7AAD staining are much clearer than those obtained using propidium iodide (PI).  Using flow cytrometry stained and unstained, transfected and non-transfected populations can be separated much more easily.

How long have you been able to observe GFP expression in primary Human Keratinocytes?

GFP expression has been observed for up to one week as measured by light and fluorescence microscopy.

In my Nucleofection Experiment I see lower expression with my IRES construct in comparison with you pmax-GFP control. Do you have any information about the different expression profiles?

The original attenuation of the IRES sequence was performed to allow for a greater difference between the expression levels of the upstream gene of interest and the downstream reporter gene. If the downstream reporter gene, the product of less than...

What are the best time points for analysis of my siRNA experiments?

As the stability and half-life of various mRNAs and their protein products varies, it is important to empirically determine the best time points for assessing target knockdown. For example, it has been documented that in mammalian cells, mRNA...

How often do mycoplasma contaminations occur in cell cultures?

A conservative estimate states that between 15-35% of all continuous cell cultures are contaminated with mycoplasma (Drexler HG, Uphof CC; 2002), some estimates are even higher (up to 80 % in some countries (Koshimizu K, Kotani H; 1981).

Do I need a special incubator for cultivation of insect cells ?

Insect cells are cultivated at lower temperatures than E.coli or mammalian cells (S2 cells at 24°C, Sf9 cells at 28°C).Therefore they need a dedicated incubator with appropriate temperature.

Can I co-transfect oligonucleotides and plasmids with the Nucleofector® Technology?

Yes. As the same protocol applies for any nucleic acid substrate (vectors or oligonucleotides) you can easily perform co-transfections either for transfection control or rescue experiments.

Can I use SDS as a detergent for a positive control if I do cytotoxicity assay with ToxiLight?

No. SDS has a negative influence on Adenylate Kinase (AK) activity. SDS is a cationic detergent, it is a very powerful protein denaturing agent, you will be aware of its use in SDS-PAGE for those reasons. ToxiLight™ works by measuring the activity of...

Which promoter can be recommended for transient gene expression in mammalian cells ?

Vectors with a CMV promoter typically work fine for transient protein expression in mammalian cells, like HEK293, CHO or NSO. For insect cells, like Sf9 or Sf21, vectors with insect promoters are required. In HEK293E (EBNA) cells, the use of...

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