BIOTECHNOLOGY 83
with 0.4µg pmaxGFP Vector using Nucleofector Solution SE, SF or SG and 31 different programs (15 of which are shown). 5 x 104
seeded and transfection efficiency was analysed 24 hours post transfection by flow cytometry (A). Viability was determined with the ViaLight Plus BioAssay Kit (B). Cell viability is expressed as per cent viability compared with the non-transfected no program controls.
cells was determined using flow cytometry (FACSCalibur, Becton Dickinson) according to standard procedures. Cell viability was determined using the ViaLight Plus BioAssay Kit (Lonza, cat. no. LT07-321), according to the protocol. Cell viability is expressed as percentage viability compared to the non-transfected no program controls.
Results Using the 4D-Nucleofector System in combination with the 96-well Shuttle Add-on, we found that the human prostate carcinoma cell line DU 145 can be easily transfected. Best results were obtained with Nucleofector Solution SE and Program CA-137, resulting in a transfection efficiency of 78 per cent (Fig. 2A) with a cell viability of 55 per cent (Fig. 2B). Tese results show that efficient Nucleofection Conditions for
transfected cells were
Fig. 2. First optimisation round for the human prostate carcinoma cell line DU 145 using the 4D-Nucleofector System in combination with the 96-well Shuttle Add-on. 2 x 105
cells were transfected
cancer cell lines can often be easily determined in a single experiment.
For the human colorectal adenocarcinoma cell line COLO 205, the initial optimisation experiment achieved maximal transfection efficiencies of ~30 per cent (Fig. 3A) with program-dependent cell viabilities (Fig. 3B). Tus, we performed two further program fine-tuning rounds and identified Nucleofector Program DP-113, in combination with Nucleofector Solution SG and a cell seeding density of 1 x 105
cells in 96-well, as
the best selections under the given experimental conditions (data not shown). However, transfection efficiency still remained below 30 per cent with a cell viability of 50 per cent.
In order to improve transfection efficiency and viability, further optimisation experiments were performed. Incubation of COLO 205 cells for 10 minutes in Nucleofector Solution post transfection increased transfection efficiency and cell viability.
An additional increase of transfection efficiency was observed when increasing the
amount of plasmid DNA per reaction. Cell viability remained above 60 per cent compared to the no program control with DNA amounts up to 1µg per reaction. With a higher DNA concentration, a drop in cell viability was observed.
One important feature of the 4D-Nucleofector X Unit is the transferability of conditions from the 20µl Nucleocuvette Strips (for low cell numbers and higher throughput) to the 100µl single Nucleocuvette Vessels (for higher cell numbers).
For the latter, the same Nucleofection Conditions can be applied using a five times higher cell number and substrate amount.
Summary Excellent transfection efficiencies of up to 99 per cent combined with high cell viability can be obtained with the 4D-Nucleofector System for different adherent and suspension cancer cell lines.
With the 96-well Shuttle Add- on, six 20µl Nucleocuvette Strips can be processed in parallel enabling screening applications as well as offering a convenient, effective and time-saving
Fig. 3. First optimisation round for the human colorectal adenocarcinoma cell line COLO 205 using the 4D-Nucleofector in combination with the 96-well Shuttle Add-on. 2 x 105
pmaxGFP Vector using Nucleofector Solution SE, SF or SG and 31 different programs (15 of which are shown). 5 x 104
transfection efficiency was analysed 24 hours post transfection by flow cytometry (A). Viability was determined with the ViaLight Plus BioAssay Kit (B). Cell viability is expressed as per centage viability compared to the non- transfected no program controls.
approach when optimising Nucleofection Conditions for multiple cells lacking a ready-to- use Optimised Protocol.
In many cases, the optimal Nucleofection Conditions can already be determined during the first optimisation round. Te same protocol can be applied for 100µl and 20µl transfection volumes, allowing the transfection of variable cell numbers.
For more information ✔ at
www.scientistlive.com/eurolab The authors are Jenny Schroeder,
Ludger Altrogge, Elke Lorbach, Sabine Schaepermeier, Meike Weigel, Gina
Andretta-Beu, Stefanie Buesch, Tamara Grabeck, Alexandra Krumnow, Sonja Spicker and Andrea Toell who are with Lonza Cologne GmbH, Koeln, Germany.
Srinivasan Kokatam, Sampada Kallol and Preeti Kapoor are with Lonza India PVT Ltd, Hyderabad, India
www.lonza.com
www.scientistlive.com transfected cells were seeded and cells were transfected with 0.4µg
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