Data Type


Category

+ Show All

Research Area

+ Show All
609 results sorted by

Is it possible to use frozen primary CD34+ hematopoietic stem cells for Nucleofection?

Yes. For cryopreserved PBMCs or enriched CD34 populations, we recommend culturing the thawed cells 1-2 hours in culture medium before Nucleofection™. Any further enrichment procedure after thawing is not recommended. Viability of frozen nucleofected...

What kind of buffer do you recommend for resuspending my siRNA?

Please use the buffer that is recommended by your siRNA manufacturer.

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

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

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.

The animal facility which isolates my hepatocytes is at a different location, should I be concerned about transport time?

Isolated hepatocytes can be transported in Krebs-Henseleit Buffer or supplemented William's E Medium. Long transport time for isolated hepatocytes, (in excess of one hour) can cause a decrease in efficiency of Nucleofection™, but does not seem to...

How much DNA can I use when transfecting hepatocytes with the standard Nucleofector?

We have used up to 6 µg of DNA per 100 µl reaction with no deleterious effect.

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 Nucleofection™....

Is it more difficult to detect DsRed by FACS than GFP?

Yes, DsRed is much less bright than GFP and is more difficult to detect by FACS and microscopy because it bleaches quickly.

Can I use the pmaxGFP control plasmid with insect cells such as S2 cells or SF9 cells?

The pmaxGFP™ Vector provided in our Nucleofector™ Kits is not expressed in insect cells. We strongly recommend an insect expression vector encoding a fluorescent protein or lacZ reporter as a positive control for your experiments [e.g. Novagen™’s...
PAGE 4