Optimization strategy - Nucleofector^® Technology 

Nucleofection conditions shared by other users

In addition to data generated by Lonza, our comprehensive Knowledge Database also contains data on Nucleofection^® Conditions that were shared by other users and which have provided sufficient results for their purposes. You may apply these conditions to your cell type of interest, but please be aware that a more comprehensive optimization (see details below) may lead to better results for your specific cell clone or source. 

Using pmaxGFP™ Positive Control for transfection optimization 
 

Regardless of what type of substrate you ultimately want to transfect for your application (plasmid, siRNA, RNPs, protein or chemical compound), we always recommend using our pmaxGFP™ Vector for optimization. Once Nucleofection conditions are determined with the pmaxGFP™ Vector, they can be used with your specific substrate (e.g. plasmid DNA, mRNA, siRNA, shRNA vector, peptide, ribonucleoprotein (RNP)). Using the pmaxGFP™ Vector for optimization eliminates a number of variables, such as the nature and quality of preparation of your substrate and the timing of analysis.

Fine-tuning of transfection results (optional)  

If results of your optimization experiment are not satisfactory yet, there is always the option to further fine-tune results. For the 4D-Nucleofector^® X Unit, 4D-Nucleofector® 96-well Unit, or the 384-well Nucleofector^® System, this can be achieved with the aid of our fine tuning matrix.  For platforms that do not offer such a matrix (4D-Nucleofector^® Y Unit or Nucleofector^® 2b System), or for additional advice, please contact our Scientific Support Team.

Option 1: Performing a cell group-specific optimization using Basic Protocols

We provide a cell group-specific optimization protocol for a number of primary cell groups. Those so-called Basic Protocols refer to a pre-selected set of Primary Cell Nucleofector^® Solutions and a limited set of Nucleofector^® Programs. Basic Protocols are available for the cell groups listed in the table and are suited for various mammalian species, e.g. human, mouse or rat.

Option 2: Performing a full optimization using Optimization Protocols

We offer optimization protocols for primary cells for most of our Nucleofector^® platforms. For the Nucleofector^® I/II/2b System, we do not have an optimization protocol for primary cells due to the large number of different cell type specific solutions. Instead, we offer cell group-specific optimization protocols – see table.
 
Using our pmaxGFP™ Vector as positive control, a set of Nucleofector^® Solutions is tested in combination with a pre-selected set of programs plus controls. Especially with our Nucleofector^® Platforms that offer small scale multi-well formats (e.g. 4D-Nucleofector^® X Unit or 4D-Nucleofector^® 96-well Unit), such optimizations are straightforward, saving time and material. Established conditions are transferable between different platforms and scales (384-well Nucleofector^® System ↔ 4D-Nucleofector^® 96-well Unit and 96-well Shuttle^® Add-On ↔ 4D-Nucleofector^® X Unit ↔ 4D-Nucleofector^® LV Unit).

Table: Full Optimized Protocols

Optimization of Nucleofection® Conditions

The Cell Line Optimization Kit as well as the Primary Cell Optimization Kit are working with a program matrix comprising 15 Nucleofection® Programs for a first optimization. In most cases you will receive good results by applying this matrix only. However for maximum performance and depending on your desired outcome a fine tuning to reach higher efficiency or higher viability is recommended. The fine tuning is ideally performed in the 16-well Nucleocuvette® Strip of the 4D-Nucleofector® X Unit or in the 4D-Nucleofecotr® 96-well Unit. To do this:

• Select the best solution and the best program(s) from the first optimization round (for example DN-100)
• Use our fine tuning matrix to select alternative programs leading to higher viability (for example DH-100) or better efficiency (for example ER-100)