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Simple Optimization Protocol for Difficult to Transfect Cells Using FuGENEĀ® HD Reagent
Sponsored,vendor-submitted protocol   Sponsored by Roche    Published in BioTechniques Protocol Guide 2008 (p.33)

Some cell lines are considered difficult to transfect because the correct parameters for transfection of that cell line are not known. The reagent:DNA ratio must be tested because laboratories may quantify DNA amounts slightly differently. The amount of complex added is an equally critical factor; too much complex may result in cytotoxicity, and too little complex may result in suboptimal expression. When testing new cell lines, we use a simple protocol which provides comparisons between four critical parameters:

  • Ratio of µl of reagent to µg of DNA

  • Amount of complex added to the cells

  • Length of time for complex to form

  • Cell density

Day 1

  1. Plate the cells in two 96-well plates at two different densities, using antibiotic-free complete medium. These are the cell plates.

Day 2

  1. Prepare the transfection complex in serum-free medium:

    1. Calculate volume needed. Prepare diluted plasmid at 20 µg/ml.

    2. Form complexes in an empty 96-well round bottom plate; this plate is the transfection complex plate. Complexes are formed on this plate; 2 wells contain controls:

      • The reagent control (top) well contains 150 µl serum-free medium and 12 µl FuGENE® HD Transfection Reagent.

      • The plasmid control (bottom) well contains 150 µl diluted plasmid.

      • The six complex wells (middle) each contain 3 µg of diluted plasmid in 150 µl medium and 12, 10.5, 9, 7.5, 6, and 4.5 µl FuGENE® HD Transfection Reagent, respectively. This produces six different ratios of reagent to DNA.

  2. As soon as the last complex has been formed, remove the cell plates from the incubator. Gently add different amounts of complex to the wells on the left side of each cell plate, such that five different complex amounts are added to each of the six complex ratios, and each row on the plate corresponds to one of the six different complex ratios from the complex plate. The same plate layout is used for both cell plates.

  3. Return the cell plates to the incubator and leave the transfection complex plate at room temperature.

  4. Wait 15 minutes, then repeat the complex addition, this time in the wells on the right side of each cell plate.

  5. Return the cell plates to the incubator until time of assay (24-72 hours depending upon gene expressed).

  6. Prior to assaying the cells for expression of the transfected gene, visually inspect the cells. Figure 1 shows the data from an experiment in which only two amounts of complex were added to the plates; the protocol is easily modified to suit individual needs.

Figure 1. (Click to enlarge)

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