What points should I consider to achieve optimal transfection with a lipid-based transfection reagent?

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Here are some points to consider:

1. Select the cationic lipid reagent that is likely to result in highest transfection efficiency for your cell type. Please refer to the Transfection Reagent Selection Guide (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html) to make the right choice.
2. Optimize the 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 may contain components that could interfere with complex formation. We recommend using Opti-MEM™ I Reduced-Serum Medium for optimal complex formation. However, serum-free DMEM or serum-free RPMI 1640 Medium can be used, keeping in mind that the efficiency of complex formation may not be as high as with Opti-MEM™ I Reduced-Serum Medium.
4. Do not use antibiotics, EDTA, citrate, phosphate, chondroitin sulfate, hyaluronic acid, dextran sulfate, or other sulfated proteoglycans in the medium used to prepare the DNA-cationic lipid reagent complexes.
5. Cell density should be from 50% to 80% confluency at the time of transfection (for Lipofectamine™ 2000, we recommend >90% confluency). Cells should be in the mid-log growth phase. For better consistency of results between transfection experiments, it would be best to accurately count your cells with a hemocytometer or with the Countess™ II FL Automated Cell Counter (Cat. No. AMQAF1000).
6. Make sure that the promoter-enhancer of the transfected DNA is compatible with the target cell type.
7. Do not use a cationic lipid reagents that has been frozen or stored in a section of the refrigerator where the temperature is below 4 degrees C.
8. Include a positive control for the transfection assay (for example, Cat. No. A14146 for plasmid DNA transfection and Cat. No. 14750100 for siRNA transfection).

Also, please take a look at the tips outlined here (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/troubleshooting-transfection-experiments.html).

Answer Id: E8989

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What controls do you recommend that I use when performing a transfection?

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In general, our recommended controls include:

- Cells only
- Cells + DNA or RNAi only
- Cells + lipid reagent only
- Cells + GFP plasmid positive control

These controls are important as they check cell health, reagent toxicity, and reagent function simultaneously.

Answer Id: E8990

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Are cell density (% confluency) and passage number important considerations for transfection?

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Yes. Cell density will affect transfection performance. Lipofectamine™ 3000, Lipofectamine™ 2000, and Lipofectamine™ LTX/PLUS provide excellent transfection performance at confluencies between 70 and 90%, while some toxicity may be observed at confluencies lower than this. Lipofectamine™ RNAiMAX works best at confluencies between 60 and 80%.

Passage number may affect transfection experiments. We recommend consistent splitting and plating of cells. Excessive numbers of passages may decrease transfection performance. We do not recommend splitting cells for more than 20-30 passages. If transfection performance declines and cells have been in culture for a long time or excessively/improperly passaged, we recommend that you restart your cultures with a new vial of cells from liquid nitrogen. Please refer to the Gibco™ Cell Culture Basics handbook (https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics.html) for proper guidelines for culturing and passaging cells.

Answer Id: E8962

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I don't see a cell line-specific transfection protocol. What conditions should I try?

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If you do not find a cell line-specific protocol or if the protocol does not perform as expected, we recommend testing the conditions described in the protocol supplied with the product to determine the optimal protocol. Successful transfection depends on the cell type, amount of lipid, cell health, passage number, and cell density at the time of transfection. Each of these factors may differ slightly from lab to lab, and require additional optimization of the protocol to achieve the same result.

Answer Id: E8992

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Can I use the same amount of any transfection reagent for different cell lines?

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No. The transfection efficiency is highly dependent on the amount of reagent used per well and may be different between reagents. Please consult the product information that is provided with the transfection reagent for optimal use. The protocol that is supplied with the product will provide you with an optimal range of transfection reagent to use per well. During product development, this range was determined to work well across a variety of cell lines. If you are still not achieving the performance you desire in your particular cell line, further optimization may be necessary.

Answer Id: E8963

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How do I choose between Lipofectamine™ 2000 and Lipofectamine™ LTX?

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Lipofectamine™ LTX and Lipofectamine™ 2000 are both recommended for plasmid DNA transfections. However, if you notice increased cytotoxicity with Lipofectamine™ 2000, Lipofectamine™ LTX is the preferred reagent due to significantly reduced cytotoxicity levels and improved transfection performance in a number of hard-to-transfect cell lines.

Answer Id: E4997

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Which lipid transfection reagent would you recommend trying for my cell line?

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We recommend using Lipofectamine™ 3000 Reagent or delivery of plasmid DNA, Lipofectamine™ MessengerMAX™ Reagent for delivery of mRNA or short oligos, and Lipofectamine™ RNAiMAX Reagent for delivery of siRNA/miRNA.

Answer Id: E8993

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I am working with well sizes different from those specified in your protocol. How do I scale up or scale down my transfection reaction?

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Each of our transfection reagent protocols provides a table for scaling up or down transfections. Please consult the specific manual for details.

Answer Id: E8964

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Is it necessary to use serum-free media during lipid transfection?

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Not in all cases. What is essential is to form the lipid:nucleic acid complex in the absence of serum, because proteins can interfere with complex formation. Once the complexes are formed, they can be added to cells in serum containing medium. For optimal results, with Lipofectin™ perform transfection in medium without serum.

Answer Id: E3131

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Why are my transfections not reproducible?

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Please review these possible causes for non-reproducible transfections, along with suggested solutions (http://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/troubleshooting-transfection-experiments.html#3). Also, we recommend setting up one master mix of the DNA/lipid complex for the number of transfections planned to reduce multiple pipetting errors. When adding your complexes, we recommend changing tips between wells since re-used tips could bring carryover, especially for the 96- or 384-well format with small-volume formats.

Answer Id: E8983

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Why is my transfection not reproducible?

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In general, transfection efficiency will show some degree of variability from one transfection to another, no matter how hard one tries to control the parameters of transfection. Keep all transfection parameters, such as cell confluency, passage number, and phase of growth, consistent between transfections. If possible, thaw fresh cells. To minimize the effect of transfection variability, one can use an internal reference control such as beta-galactosidase or luciferase. Co-transfect the expression plasmid with the reference plasmid and assay for the activity of beta-gal or luciferase.

Answer Id: E8965

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Can antibiotics be used in the medium during transfection?

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Answer

Yes, antibiotics (penicillin-streptomycin) can be used in the medium during transfection. We have compared transfecting cells in medium with and without antibiotics in multiple cell lines, assessed both the transfection efficiency and toxicity, and found no difference. For certain cell types that are sensitive to transfection or have toxicity issues, omitting antibiotics may improve results, however. For stable transfections, wait at least 72 hours after transfection before adding selective antibiotics.

Answer Id: E8998

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Are lipid transfection reagents fluorescent?

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Answer

The intrinsic fluorescence of Lipofectamine™ 2000 and Lipofectamine™ under FITC conditions was examined and the results were as follows:
- Lipofectamine™ 2000 in serum-free conditions (with or without DNA) - weak whitish fluorescence.
- Lipofectamine™ 2000 in serum-containing conditions (with or without DNA) - medium white and red fluorescence.
- Lipofectamine™ & Lipofectamine™ PLUS (with or without DNA) - orange fluorescence.
- We haven't seen any significant green fluorescence attributable to the Lipofectamine™.

These observations were made after transfection of CHO-K1 cells and by examining the live cells in PBS. Other lipid transfection reagents were not studied extensively on this issue, but it is very likely they will produce fluorescence in transfected cells.

Fluorescence from various media components may be where the problem lies. For example, riboflavin apparently fluoresces in the green wavelength and thus DMEM, which has a high riboflavin content, can be problematic in fluorescence experiments. Ham's F12 medium is 10x lower in riboflavin and is more suitable for fluorescence work.

One possible solution is to perform fluorescence imaging in PBS, rather than in culture medium. Also, check to make sure the cells are healthy and intact. Lysed cells or cells under stress may generate autofluorescent products.

Answer Id: E4451

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Is my lipid (Lipofectin™, Lipofectamine™, DMRIE-C, Lipofectamine™ 2000, Oligofectamine™, Cellfectin™ II) supposed to be cloudy?

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Answer

It is normal to see some turbidity and cloudiness in DMRIE-C, Cellfectin™ II and Lipofectamine™ 2000, and the others should be clear. Lipid transfection reagents are sensitive to low temperature; if you put them at temperature below 4 °C they will precipitate or even freeze and lower the efficiency. Most lipids will go cloudy and precipitate upon freezing, and may not be active anymore.

Answer Id: E4000

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What is the shelf life of the lipid transfection reagents?

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Answer

Stored at 4°C in a sealed container, the lipids are stable for 12 months. Do not freeze the lipids. At 4°C with long term storage, due to evaporation concentration of lipid may vary, please briefly spin the contents before use. Add less lipid if you start noticing toxicity.

Answer Id: E3132

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