We generally produce adenoviral stocks in 293A cells using the following optimized transfection conditions. The amount of adenovirus produced using these recommended conditions is approximately 10 ml of crude viral lysate with a titer ranging from 1 x 10e7 to 10e8 pfu/ml. We use Lipofectamine 2000 for transfection. If using another transfection reagent, use the manufacturer’s recommendations.

Tissue culture plate size: 6-well plate (one well per adenoviral construct) Number of 293A cells to transfect: 5 x 105 cells Amount of Pac I-digested pAd-DEST expression plasmid: 2 ug Amount of Lipofectamine 2000 6 ul

293A cells should be plated 24 hours prior to transfection in complete medium, and should be 90-95% confluent on the day of transfection. Make sure the cells are healthy at the time of plating.

Incubate Lipofectin Reagent in OPTI-MEM I Medium (or other medium without serum) for 30 minutes prior to adding DNA to help improve the transfection efficiency upto 3-fold.

Getting a cell transfected and starting a productive viral infection are two different things. If only one or two cells in your lawn are producing virus, it will take quite a while for that to be visible to the naked eye - most people would give up before that happens. Transfection efficiency is correlated with the start of virus production simply in that the more cells you get DNA into, the more chance you have of seeing virus production within the first week or two. Lower transfection efficiencies will eventually produce virus but you have to wait a long time to see it.

In general, efficiencies will show some degree of variability from transfection to transfection, no matter how hard one tries to control the parameters of transfection. Keep all transfection parameters, such as confluency, passage number, and phase of growth, consistent between transfections. If possible, thaw fresh cells. To minimize variability from transfections, you can use internal reference control for example B galactosidase or luciferase. Cotransfect your expression plasmid with the reference plasmid and assay for the activity of B-gal/luciferase. See FOCUS 17.2, 60.

It generally yields better transfection activity (as measured by protein expression) than all other lipids in a majority of the cell lines tested. It includes a streamlined, simple protocol where the complexes are added directly to cells without changing media. This lends itself to high throughput applications. It works very well in the presence or absence of serum. Examples of cells that show the highest transfection efficiency with LIPOFECTAMlNE 2000 include 293 F, 293 H, BE(2)C, CHO-K1, CHO-S, COS-1, COS7-L, Human Primary Fibroblasts, Ht-29, HT-1080, MDCK, MRC-5, PC12 and Vero.

1. Too much DNA or lipid used. Do a dose-response curve to determine the optimal amount of DNA and cationic lipid reagent. DNA or cationic lipid alone generally has a minimal effect on cell growth. Consult the technical manual for optimization protocols for a specific lipid.

2. Do not use chloroquine, penicillin, or streptomycin during transfection because cationic lipid reagents make cells more sensitive.

3. Do a dose-response curve to determine the optimal number of cells/transfection.

4. Use OPTI-MEM I Medium during transfection. Reduce or omit the number of washes in serum-free medium. Use 5% to 10% serum in the transfection medium. Be sure to form complexes in the absence of serum.

5. Do not vortex or agitate cationic lipid reagents excessively; this may form cationic lipid reagent peroxides. 6.For stable transfection, allow at least 48 h for cells to express resistance before adding selective antibiotic.

1. Select the cationic lipid reagent likely to result in highest transfection efficiency for your cell type. See the references listed in the Transfection Collection on our web site.

2. Optimize cationic lipid reagent and DNA amounts. The most important parameter after the condition of the cells is the ratio of lipid to DNA.

3. Do not use serum during complex formation. Serum contains lipids which will interfere with complex formation.OPTI-MEM I Reduced-Serun Medium or DMEM (use either without adding serum during complex formation) is good media for complex formation.

4. Do not use antibiotics, EDTA, citrate, phosphate, RPMI, chondroitin sulfate, hyaluronic acid, dextran sulfate, or other sulfated proteoglycans in the medium used to prepare DNA-cationic lipid reagent complexes.

5. Cell density should be 50% to 80% confluent at the time of transfection (for Lipofectamine 2000, we recommend >90% confluency). Cells should be in the mid-log growth phase.

6.Make sure the promoter-enhancer of the transfected DNA is compatible with the target cell type.

7.Do not use cationic lipid reagent that has been frozen or stored in a section of the refrigerator where the temperature is <4 degree.

8.Include a positive control for the transfection assay.

 Optimization of transfection conditions is essential for the highest-efficiency transfections and the lowest toxicity. The conditions that should be optimized include Lipofectamine 2000 and DNA concentrations, and cell number. It is recommended that conditions be tested and optimized for cell lines, which do not have cell specific protocols available.

To optimize the amount of Lipofectamine 2000 reagent, start with cells at >90% confluency and 0.8-1.2 µg DNA for 24-well plates. With cell number and DNA concentration held constant, vary the amount of Lipofectamine 2000 Reagent to determine the optimal concentration (usually 1.5-3 µl). The cell number and amount of DNA can also be optimized. It is possible to minimize the effect of transfection on cell growth and viability by increasing the number of cells plated per well or by decreasing either Lipofectamine 2000 reagent or DNA concentrations. With careful optimization, this can be achieved with little impact on the level of transgene expression.