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What stock concentration of Propidium Iodide does Lonza use for FACS analysis after Nucleofection®?

We use a stock concentration of 100 µg/ml in PBS. This is diluted 1:300 – 1:400 into the sample.

Do you have a protocol for the Nucleofection® of Plasmodium berghei using the Nucleofector® Systems

Nucleofector I/II/2bWe have excellent data for the Nucleofection™ of Plasmodium berghei from the group Chris J. Janse and Andrew P. Waters from the University of Leiden. Their protocol describes a transfection efficiency of 10-3 – 10-4. With this...

In your optimized Nucleofection® Protocols for MDA-MB-231 and MDA-MB-468, you say to culture the cells without CO2. Why?

The media recommended for these cells is Leibovitz's L-15 medium which does not contain sodium bicarbonate to act as a buffer for the carbonic acid that would normally form in the presence of CO2. Furthermore, if the cells are cultured in Leibovitz's...

Do you have any siRNA Nucleofection® results using concentrations lower than 50nM?

Yes. Please click on the attached Technical Reference Guide: "Designing an RNAi Experiment Using Nucleofection®". On page 3 you can find a table with some examples.

For hepatocytes, is plating density a concern post Nucleofection®?

Yes, the optimized protocols for the standard Nucleofector® recommend the use of 6 well plates. Plating into a 12-well plate will pose a problem as less cells will attach at high densities.

Four hours after Nucleofection®, I can see the hepatocytes have attached to the well, but the morphology does not look correct, should I be concerned?

There is no reason for alarm. The hepatocytes may not exhibit normal morphology a few hours after Nucleofection® but by 24 hours post Nucleofection®, morphology should be normal. Remember to perform a fluid change about 4 hours post...

Is there a difference between, for example, the programs C-20 and C-020?

No, the programs C-20 and C-020 are identical regarding their electrical parameters. The letter minus a two-digit number is the nomenclature of the Nucleofector™ I. Since we have launched the Nucleofector™ II we changed it to a letter minus a three...

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.

Why do plasmids which contain IRES sequences often have lower transfection efficiency?

In general, there is not a problem using IRES plasmids with Nucleofection®, with one important caveat. The levels of expressed protein for the first and second genes will not be identical, and this can create problems with analysis and interpretation...

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.

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