PLUS™ Reagent

PLUS™ Reagent is a proprietary reagent for pre-complexing DNA that enhances cationic lipid-mediated transfection of DNA into many cultured eukaryotic cells. Use PLUS™ Reagent to enhance transfection results with Lipofectamine® LTX Reagent, Lipofectamine® Reagent, Lipofectin® Reagent, Cellfectin® Reagent, and Oligofectamine™ Reagent.

Guidelines for Transfection

• PLUS™ Reagent enhances transfection mediated by cationic lipids; do not use PLUS™ Reagent on its own.
• PLUS™ Reagent may enhance vector-based RNAi experiments, but is not recommended for siRNA and Stealth™ RNAi transfections.
• The efficacy of PLUS™ Reagent depends on the cell line; test each cell line individually.
• Visit www.lifetechnologies.com/transfection or contact Technical Service for specialized transfection protocols (including cell-type specific advice).
• Refer to the manual provided with your transfection reagent for additional transfection guidelines.
• Some serum-free media (such as CD 293 Medium, 293 SFM II, and VP-SFM) inhibit cationic lipid-mediated transfection. Test media for compatibility with transfection reagent before use.

Use this procedure to transfect DNA into mammalian cells in a 24-well format using Lipofectamine® LTX Reagent and PLUS™ Reagent. For other formats, see Scaling Up or Down Transfections. All amounts are given on a per well basis. Use this procedure as a starting point; optimize transfections as described in Optimizing Transfections.

Note: Read Important Guidelines for Transfection and the Lipofectamine® LTX Reagent manual before starting.

1. Adherent cells: One day before transfection, plate cells in 500 μl of growth medium so that the cells will be 50-80% confluent at the time of transfection. Suspension cells: Just prior to preparing complexes plate 200,000-500,000 cells in 500 μl of growth medium.


2. For each transfection sample, prepare complexes as follows:
   
   
   • a. Dilute 500 ng plasmid DNA in 100 μl Opti-MEM® I Reduced Serum Medium without serum. Mix gently.
   
   
   • b. Mix PLUS™ Reagent gently before use, then add 0.5 μl PLUS™ Reagent directly to the diluted DNA. Mix gently and incubate for 5-15 minutes at room temperature.
   
   
   • Mix Lipofectamine® LTX Reagent gently before use, then add 1.25 μl directly to the diluted DNA. Mix gently.
   
   
   • Incubate for 30 minutes at room temperature. Complexes are stable for 6 hours at room temperature.


3. Add the ~100 μl DNA-Lipofectamine® LTX complexes to the well containing cells. Mix gently by rocking the plat  back and forth.


4. Incubate the cells at 37°C in a CO₂ incubator for 18-48 hours prior to testing for transgene expression. Medium may be changed after 4-6 hours.

 

Passage cells at 1:10 (or higher dilution) into fresh medium 1 day after transfection with either protocol. Add selective medium (if desired) the next day.  Several days or weeks of selection are still required for stable expression.

Use this procedure to transfect DNA into mammalian cells in a 24-well format using Lipofectamine® Reagent and PLUS™ Reagent. For other formats, see Scaling Up or Down Transfections. All amounts are given on a per well basis. Use this procedure as a starting point; optimize transfections as described in Optimizing Transfections.

Note:   Read Important Guidelines for Transfection on page 1 and the Lipofectamine®  Reagent manual before starting.

1. The day before transfection, plate cells in 24-well plates so that they are 50-80% confluent the day of transfection. At the time of plating and during transfection, avoid antibiotics.


2. Pre-complex the DNA with the Plus™ Reagent: Dilute 0.4 μg DNA into 25 μl dilution medium without serum (Dulbecco’s Modified Eagle Medium or similar medium). Mix Plus™ Reagent before use. Add 4 μl Plus™ Reagent to diluted DNA, mix again, and incubate at room temperature for 15 minutes.


3. Dilute 1 μl Lipofectamine®  Reagent into 25 μl dilution medium without serum in a second tube; mix.


4. Combine pre-complexed DNA (from step 2) and diluted Lipofectamine® Reagent (from step 3); mix and incubate for 15 minutes at room temperature.


5. While complexes are forming, replace the medium on the cells with 0.2 ml of cell growth medium without serum. Note:   It is possible to include serum in the cell growth medium at this step.


6. Add the DNA-Plus™-Lipofectamine® Reagent complexes to each well of cells containing fresh medium. Mix complexes into the medium gently; incubate at 37°C at 5% CO₂ for 3 hours.


7. After the 3 hours incubation, increase volume of medium to normal volume; add serum to bring the final concentration to that of normal growth medium. If necessary to maximize cell growth, replace the medium containing the complexes with fresh, complete medium after 3 h incubation or the day after transfection.


8. Assay cell extracts or stain cells in situ for reporter gene activity 24-48 hours after the start of transfection, depending on cell type and promoter activity.

Plus™ Reagent can also be used to enhance transfections with Lipofectin® Reagent, Cellfectin® Reagent, and Oligofectamine™ Reagent. Use the procedure described in Enhancing Transfections with Lipofectamine® Reagent, but replace Lipofectamine® Reagent with your transfection reagent. Use the cell density and additional transfection guidelines recommended in the manual provided with your transfection reagent.

To obtain the highest transfection performance, vary the amounts of DNA, PLUS™ Reagent and lipid. Consider varying cell density. For optimizing transfections with Lipofectamine® LTX Reagent and PLUS™ Reagent, we recommend testing ratios of DNA (in μg) to PLUS™ Reagent (in μl) of 1:0.5; 1:1 and 1:2. Alternatively, keep the DNA:PLUS:LTX ratios constant but vary the amount of complex added to each well. Refer to the manual provided with your transfection reagent for additional suggestions.

Note: Visit www.lifetechnologies.com/transfection for cell-specific transfection protocols.

To transfect cells in different tissue culture formats, vary the amounts of lipid, DNA, cells, medium and PLUS™ Reagent used in proportion to the relative surface area of the culture vessel. If volumes of lipid or PLUS™ Reagent are too small to dispense accurately (<0.5 μl), predilute in Opti-MEM® I Reduced Serum Medium or dilution medium. Discard any unused diluted reagents.

PLUS™ Reagent is tested for the following aspects:

• For absence of microbial and fungal contamination with blood agar plates, Sabaraud dextrose agar plates, and fluid thioglycolate medium.
• Functionally by transfection of human fibroblasts with a reporter plasmid and Lipofectamine® Reagent.

This product is covered by Limited Use Label License No.5: Invitrogen Technology (see the Life Technologies catalog or our website, www.lifetechnologies.com). By the use of this product you accept the terms and conditions of all applicable Limited Use Label Licenses.