Notes & Tips Optimization of the transfection of human THP-1 macrophages by application of Nunc UpCell technology Marten B. Maeß a , Andrea-Anneliese Keller a , Knut Rennert b , Alexander Mosig b,c , Stefan Lorkowski a,⇑ a Institute of Nutrition, Friedrich Schiller University Jena, 07743 Jena, Germany b Department of Molecular Hemostaseology, Jena University Hospital, 07743 Jena, Germany c Integrated Research and Treatment Center, Center for Sepsis Control and Care (CSCC), Jena University Hospital, 07747 Jena, Germany article info Article history: Received 2 December 2014 Accepted 23 December 2014 Available online 4 February 2015 Keywords: THP-1 macrophages Transfection siRNA Detachment abstract We have established an electroporation protocol for transfection of premature adherent human THP-1 macrophages using Lonza Nucleofector technology. For efficient electroporation, detachment of adherent cells is necessary. We tested the Nunc UpCell product line of Thermo Fisher Scientific, which achieves detachment by a change of ambient temperature, as an alternative to enzymatic detachment. Here we present data verifying proper cell morphology and vitality and high transfection efficiency for macro- phages cultured on UpCell plates. Appropriate macrophage behavior was confirmed by measuring mark- ers of macrophage differentiation and polarization by reverse transcription quantitative polymerase chain reaction (RT–qPCR). In conclusion, Nunc UpCell materials are a viable alternative to enzymatic detachment. Ó 2015 Elsevier Inc. All rights reserved. Macrophages are generally difficult to transfect cells. Nonproliferating, fully matured macrophages are not accessible for most nonviral transfection approaches. We previously devel- oped a transfection protocol for premature human THP-1 and pri- mary macrophages based on Lonza Nucleofection technology. This allows for transfection of macrophages with high transfection efficiencies and high cell viability [1,2]. Our protocol has been suc- cessfully applied for studies on cell signaling and cellular protein localization, among other settings [3–5]. An optimized protocol has also been established for investigating macrophage polariza- tion; in particular, Mouse T-Cell Nucleofector medium was selected as the best culture medium [6]. Nucleofection is a special- ized electroporation whose chemical and electric properties and specifications have been adjusted to achieve simultaneous permeabilization of both cell and nuclear membrane [7]. Thus, nucleic acids can immediately enter the nucleus. Because cells in suspension are needed for Nucleofection using the conventional Nucleofector 2b device, it is necessary to detach the premature macrophages. We routinely apply Accutase I [1,2]. Accutase I is a mixture of different collagenases and other proteases for gentle detachment of sensitive cells by degrading their extracellular matrix. However, enzymatic detachment approaches bring certain disadvantages in that enzymes may degrade cell surface molecules, thereby altering the cellular behavior in response to stimulation or affecting subsequent measurements such as flow cytometry and immuno cytochemistry. Alternative detachment procedures that avoid proteases are available. Different thermoresponsive poly- mers have been developed. The cell culture with surfaces coated with these polymers allows a very gentle detachment of cells by simply changing ambient temperature. The UpCell technology of Nunc is one such example. The UpCell polymers are slightly hydrophobic at 37 °C, which enables cell attachment and growth. At temperatures below 32 °C, the polymer becomes hydrophilic, thereby forming an aqueous film between cultured cells and poly- mers and resulting in a gentle detachment of cells, including their surrounding native extracellular matrix structures. Here we com- pare the performance of Nunc UpCell plates for cell detachment versus enzymatic detachment. THP-1 cells were differentiated into macrophages on UpCell (Thermo Fisher Scientific, Waltham, MA, USA) and uncoated poly- styrene (PS) 1 cell culture 6-well plates (TPP, Trasadingen, Switzerland) for 48 h with 10 ng/ml phorbol 12-myristate 13-ac- etate (Fisher Scientific, Schwerte, Germany) in supplemented RPMI 1640 medium (Sigma–Aldrich, Seelze, Germany) [6]. Detachment was performed by either change of temperature (15 min at 27 °C) http://dx.doi.org/10.1016/j.ab.2014.12.023 0003-2697/Ó 2015 Elsevier Inc. All rights reserved. ⇑ Corresponding author. Fax: +49 3641 949712. E-mail address: stefan.lorkowski@uni-jena.de (S. Lorkowski). 1 Abbreviations used: PS, polystyrene; RT–qPCR, reverse transcription quantitative polymerase chain reaction; siRNA, short interfering RNA; mRNA, messenger RNA; LPS, lipopolysaccharide; IFNc, interferon gamma. Analytical Biochemistry 479 (2015) 40–42 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio