For the transfection of primary cells and cell lines using the Nucleofector™ Technology, transfection optimization is usually not required. Lonza provides ready-to-use, cell type-specific Optimized Protocols for more than 150 cell types. In addition, our Knowledge Database contains transfection data for close to 1500 cell types. To determine if an Optimized Protocol is available for your cell type of interest, search our Knowledge Database or contact the Scientific Support Team.

However, if no cell type-specific protocol is available for your cell type of interest, transfection optimization is a simple, straightforward process. 

View the decision flow chart below to determine the best optimization strategy for your cell type.

Transfection Optimization Decision Tree  Table 2 Option A Option B Option C Scientific Support Fine tuning matrix

Option A – Performing a full optimization using Optimization Protocols

For each Nucleofector™ Platform, we offer an optimization protocol for cell lines and for most platforms, we offer an optimization protocol for primary cells. For the Nucleofector™ I/II/2b Device, 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 – refer to option B below.

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 96-well Shuttle™ Add-On), such optimizations are straightforward, saving time and material. Established conditions are transferable between different platforms and scales (384-well HT Nucleofector™ System ↔ 96-well Shuttle™ Add-On ↔ 4D-Nucleofector™ X Unit ↔ 4D-Nucleofector™ LV Unit).

Table 1: Full Optimization Protocols


  4D-Nucleofector™ X Unit 4D-Nucleofector™ Y Unit 96-well Shuttle™ Add-On HT Nucleofector™ System Nucleofector™ I/II/2b Device
Primary Cells OP-322 OP-339 OP-328 contact Scientific Support see Option B
Cell Lines OP-295 OP-339 OP-237 contact Scientific Support OP-238


Option B – Performing a cell group-specific optimization using Basic Protocols

In addition to a complete optimization as described in option A above, we also 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 (1 or 2) and a limited set of Nucleofector™ Programs. Basic Protocols are available for the following cell groups and are suited for various mammalian species, e.g. human, mouse or rat.

Table 2: Basic Protocols

  4D-Nucleofector™ X Unit 4D-Nucleofector™ Y Unit 96-well Shuttle™ Add-On HT Nucleofector™ System Nucleofector™ I/II/2b Device
Endothelial cells (mammalian) OP-320 OP-338 OP-272 contact Scientific Support OP-230
Epithelial cells (mammalian) OP-317 see Table 1 OP-231 contact Scientific Support OP-229
Fibroblasts (mammalian) OP-318 see Table 1 OP-234 contact Scientific Support OP-226
Glial cells (mammalian) OP-316 see Table 1 OP-259 contact Scientific Support OP-326
Neurons (mammalian) OP-316 OP-337 OP-259 contact Scientific Support OP-228
Smooth muscle cells (mammalian) OP-319 see Table 1 OP-260 contact Scientific Support OP-227
Stem cells (human) OP-315 see Table 1 OP-262 contact Scientific Support OP-203

Option C  – Test conditions shared by other users

In addition to data generated by Lonza, our comprehensive cell 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 using options A and B described above may provide 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, 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). 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, the 96-well Shuttle™ Add-On or the HT 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 Device), or for additional advice, please contact our Scientific Support Team.