Method

1 Wash recipient macrophages twice at room temperature in a large volume of serum-free RPMI1540 medium by centrifugation for 5-10 min each at 250-500 g.

2 Remove the supernatant fluid from the final cell pellet by aspiration.

3 Resuspend the macrophages in cold PBS to a final concentration of 3 x 106 cells/ml or higher."

4 Transfer 300 jxl of this cell suspension into an electroporation cuvette, keeping everything on ice.

5 Add plasmid DNA to the macrophage suspension to a final DNA concentration of 50 M-g/ml.

6 Tap the cuvette gently several times with an index finger to disperse the plasmid among the cells."

7 Incubate the macrophage/DNA suspension on ice for 5 min.

8 Remove the cuvettes from the ice and dry the outside.

9 Place the cuvettes in the electroporation apparatus and pulse the cells twice, using appropriate conditions.1

10 Immediately following the second pulse, dilute the cell suspension with 5 m! of RPMI 1640 supplemented with 20% FBS.

11 Plate the resulting cell suspension in a 60 mm tissue culture dish.

12 Incubate the transfected cells for 24 h at 37°C, 5-7% C02, prior to further stimulation and/or assessment of reporter gene expression,

JThe number of cells needed for each electroporation reaction will differ for individual cell populations and should be determined experimentally.

bThe concentration of plasmid DNA needed to achieve efficient transfection will vary, and should be optimized in each case.

cThe voltage and capacitance settings to use for successful electroporation will vary with each macrophage population and should be optimized in each case. The following is a successful electroporation protocol used for transient transfection of the murine macrophage cell line RAW 264.7 using the BTX 600 electroporation apparatus: 2.5 kV resistance; 400 jj,F capacitance; 24 Ohm resistance timing (R4); charging voltage set to 140 V (peak pulse voltage will be approximately 130 V). Some or all of these parameters may need to be adjusted for particular macrophage populations.

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