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Amaxa? Basic Nucleofector? Kit for Primary Mammalian Fibroblasts
For Primary Mammalian Fibroblasts
Note Cells derived from mammalian fibroblastoid tissues from various organs; adher

ent cells Mammalian fibroblasts display significant phenotypic variations due to the wide range of both species and body sites from which they may be sourced. You can determine the optimal Nucleofection? condition for your fibroblasts using the Basic Nucleofector? Kit for Primary Mammalian Fibroblasts [Cat. No. VPI-1002]. Please find some guidelines on fibroblast cell culture for Nucleofection? and on the transfection procedure using our Basic Nucleofector? Kit below. However, we recommend referring to more detailed culture protocols before you start the experiments. Having tested various fibroblastoid cell types, high transfection efficiencies could be achieved using one of the programs indicated below. If you do not attain satisfying results with your fibroblasts of interest, please contact our Scientific Support Team for further help with the optimization.

Product Description
Cat. No. VPI-1002 Size (Reactions) 25 Basic Nucleofector? Solution for Mammalian Fibroblasts 2.25 ml (2.05 ml + 10% overfill) Supplement 0.5 ml (0.45 ml + 10% overfill) pmaxGFP? Vector (0.5 ?g/?l in 10 mM Tris pH 8.0) 30 ?g Certified cuvettes 25 Plastic pipettes 25 Storage and stability Store Nucleofector? Solution, Supplement and pmaxGFP? Vector at 4°C. For long-term storage, pmaxGFP? Vector is ideally stored at -20°C. The expiration date is printed on the solution box. Once the Nucleofector? Supplement is added to the Nucleofector? Solution it is stable for three months at 4°C.

Optimization Guidelines

The initial optimization experiment is comprised of 6 reactions: 5 different Nucleofector? Programs are tested with 1 Nucleofector? Solution plus 1 control (no program). The Nucleofector? Program which turns out to be the most appropriate should be used for all subsequent transfections. A further fine tuning of the Nucleofection? condition can be performed with the help of our Scientific Support Team.
Sample 1 Program A-024 Sample 2 T-016 Sample 3 U-012 Sample 4 U-023 Sample 5 V-013 Sample 6 No program

Basic Nucleofector? Protocol for Mammalian Fibroblasts

Required Material

Note

Please make sure that the entire supplement is added to the Nucleofector? Solution. The ratio of Nucleofector? Solution to supplement is 4.5 : 1. For a single reaction use 82 ?l of Nucleofector? Solution plus 18 ?l of supplement to make 100 ?l of total reaction volume. – Nucleofector? Device – Supplemented Nucleofector? Solution at room temperature – Supplied certified cuvettes – Supplied plastic pipettes – Supplied pmaxGFP? Vector – Substrate of interest, highly purified, preferably by using endotoxin free kits; A260 : A280 ratio should be at least 1.8 – 6-well plates or culture system of your choice – For trypsinization: Reagent Pack? Subculture Reagent Kit containing Trypsin/EDTA, HEPES Buffered Saline Solution (HBSS) and Trypsin Neutralizing Solution (TNS) [Lonza, Cat. No. CC-5034]. Alternatively if cells hardly detach: Trypsin 0.5%-EDTA 0.2% – Culture medium: Please use a medium especially suited for the culture of primary mammalian fibroblasts, e.g. FGM?-2 BulletKit? [Lonza, Cat. No. CC-3132] or a different special medium recommended for your fibroblast cell type containing all required supplements. We recommend storing 40 ml aliquots of the prepared medium at -80°C. Do not use medium stored at 4°C for more than two days, as this may lead to reduced cell viability and transfection efficiency – Prewarm appropriate volume of culture medium to 37°C (1.5 ml per sample) – Appropriate number of cells (0.5 – 1 x 106 cells per sample) Minimal cell number: 2 x 105 cells (a lower cell number may lead to major in crease in cell mortality) Maximum cell number: 2 x 106

1. Pre Nucleofection?

Note

Transfection results may be donor – dependent. Culture conditions may differ between cell types. Please follow your established procedure or the supplier’s recommendations. Cell culture recommendations

1.1 1.2 1.3 1.4 1.5

Replace medium every 2 – 4 days (2 – 3 ml per 75 cm2 flask) Cells should be passaged after reaching 70 – 90 % confluency Do not use cells after passage 14 for Nucleofection? Cells should be passaged 2 – 4 days before Nucleofection? Optimal confluency before Nucleofection? 80 – 90% Trypsinization

1.6 Remove media from the cultured cells and wash cells once with HBSS; use at least same volume of HBSS as culture media 1.7 For harvesting, incubate the cells ~5 minutes at 37°C with recommended volume of indicated trypsinization reagent (please see required material)

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Basic Nucleofector? Protocol for Mammalian Fibroblasts

1.8 Neutralize trypsinization reaction with TNS once the majority of the cells (>90%) have been detached

2. Nucleofection?

One Nucleofection? Sample contains 0.5 – 1 x 106 cells 1 – 5 ?g plasmid DNA (in 1 – 5 ?l H2O or TE) or 2 ?g pmaxGFP? Vector or 30 – 300 nM siRNA (3 – 30 pmol/sample) 100 ?l Nucleofector? Solution 2.1 Please make sure that the entire supplement is added to the Nucleofector? Solution 2.2 Prepare 6-well plates by filling appropriate number of wells with 1.5 ml of supplemented culture media and pre-incubate/equilibrate plates in a humidified 37°C/5% CO2 incubator 2.3 Harvest the cells by trypsinization (please see 1.6 – 1.8) 2.4 Count an aliquot of the trypsinized cells and determine cell density 2.5 Centrifuge the required number of cells (0.5 – 1 x 106 cells per sample) at 90xg for 10 minutes at room temperature 2.6 Resuspend the cell pellet carefully in 100 ?l room temperature Nucleofector? Solution per sample 2.7 Combine 100 ?l of cell suspension with 1 – 5 ?g DNA, 2 ?g pmaxGFP? Vector (recommended for initial optimization) or 30 nM – 300 nM siRNA (3 – 30 pmol/sample) or other substrates 2.8 Transfer cell/DNA suspension into certified cuvette; sample must cover the bottom of the cuvette without air bubbles. Close the cuvette with the cap 2.9 Select the appropriate Nucleofector? Program. Please try all 5 Nucleofector? Programs initially to determine the most appropriate one for your fibroblast cell type for all subsequent experiments A-024, T-016, U-012, U-023, V-013 (A-24, T-16, U-12, U-23 and V-13 for Nucleofector? I Device) 2.10 Insert the cuvette with cell/DNA suspension into the Nucleofector? Cuvette Holder and apply the selected program 2.11 Take the cuvette out of the holder once the program is finished 2.12 Add ~500 ?l of the pre-equilibrated culture media to the cuvette and gently transfer the sample immediately into the 6-well plate (final volume 1.5 ml media per well). Use the supplied pipettes and avoid repeated aspiration of the sample

3. Post Nucleofection?

3.1 Incubate the cells in a humidified 37°C/5% CO2 incubator until analysis. Gene expression or down regulation, respectively, is often detectable after only 4 – 8 hours but ideally, cells should be left undisturbed for 24 hours

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Basic Nucleofector? Protocol for Mammalian Fibroblasts

Additional Information

For an up-to-date list of all primary mammalian fibroblasts successfully transfected with this Basic Nucleofector? Kit, please refer to: www.amaxa.com/celldatabase For an up-to-date list of all Nucleofector? References, please refer to: www.lonza.com/nucleofection-citations For more technical assistance, contact our Scientific Support Team: USA /Canada Phone: 800 521 0390 (toll-free) Fax: 301 845 8338 E-mail: scientific.support@lonza.com Europe and Rest of World Phone: +49 221 99199 400 Fax: +49 221 99199 499 E-mail: scientific.support.eu@lonza.com

Lonza Cologne AG 50829 Cologne, Germany
Please note that the Amaxa? Nucleofector? Technology is not intended to be used for diagnostic purposes or for testing or treatment in humans. The Nucleofector? Technology, comprising Nucleofection? Process, Nucleofector? Device, Nucleofector? Solutions, Nucleofector? 96-well Shuttle? System and 96-well Nucleocuvette? Plates and Modules is covered by patent and/or patent-pending rights owned by Lonza Cologne AG. Amaxa, Nucleofector, Nucleofection, maxGFP, Reagent Pack, FGM and BulletKit are either registered trademarks or trademarks of the Lonza Group or its affiliates. Other product and company names mentioned herein are the trademarks of their respective owners. This kit contains a proprietary nucleic acid coding for a proprietary copepod fluorescent protein intended to be used as a positive control with this Lonza product only. Any use of the proprietary nucleic acid or protein other than as a positive control with this Lonza product is strictly prohibited. USE IN ANY OTHER APPLICATION REQUIRES A LICENSE FROM EVROGEN. To obtain such a license, please contact Evrogen at license@evrogen.com. The CMV promoter is covered under U.S. Patents 5,168,062 and 5,385,839 and its use is permitted for research purposes only. Any other use of the CMV promoter requires a license from the University of Iowa Research Foundation, 214 Technology Innovation Center, Iowa City, IA 52242. The use of this product in conjunction with materials or methods of third parties may require a license by a third party. User shall be fully responsible for determining whether and from which third party it requires such license and for the obtainment of such license. No statement is intended or should be construed as a recommendation to infringe any existing patent. ? Copyright 2009, Lonza Cologne AG. All rights reserved DPI-1002 05/09

DPI-1002_2009-05

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