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Does the Mouse T cell protocol work for stimulated mouse T cells?

Yes. Stimulate the cells with anti CD3 and anti CD28 antibody for 24 hours. Use the optimized protocol. Often times, this will result in higher transfection efficiency (60-70% is possible). Alternatively, you can stimulate with ConA and IL-2 for 2-3...

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...

Can I apply more than one program (transfection condition) per plate with the 96-well Shuttle® System?

With the 96-well Shuttle® system, it is possible to apply up to 96 different programs (transfection conditions) per 96-well Nucleocuvette® Plate. Therefore, optimization of Nucleofection® Conditions for a particular cell line can be...

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.

What cell number do I need per well when using the 96-well Shuttle® Device or the 4D-Nucleocuvette® Stripes?

The cell number is very much dependent on the cell type you use. For high throughput Nucleofection®, we usually start with one fifth of the optimal amount recommended for the standard Nucleofection® in the 100µl cuvette. Currently the absolute...

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.

What is the amount of DNA needed per well when using the 96-well Shuttle® Device?

The amount depends on the cell type and on the DNA construct. As a rule of thumb 400 ng of DNA per well should be applied. However, for some cells or constructs even 100 ng may be sufficient.

What is the amount of siRNA needed per Nucleofection® Reaction?

When performing siRNA-mediated knockdown experiments it is advisable to conduct a dose-response (concentration) analysis to determine the minimum siRNA concentration necessary for sufficient target knockdown on the mRNA, protein, or functional level....

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.

Which reporter gene should I choose for my reporter gene experiment?

In general, all reporter genes can be used.For some applications (e.g. reporter gene experiments in various primary cells, experiments with late analysis time point due to starving and /or stimulation) a reporter gene with a longer half life than...

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