Equipment and reagents

• Humidified C02 incubator suitable for cell culture

• Sterile 6-well tissue culture plates (Becton Dickinson Lab ware)

■ Table-top centrifuge for spinning cells (Dupont/NEN)

Method

1 Add 0.625 ml of serum-free RPMI 1640 medium to each well of a 6-well tissue culture plate.

using lipophilic reagents

• Macrophage population of choice (see Chapters 1 and 2)

• Supercoiled plasmid DNA (see Protocol 4)

• Lipophilic transfection reagent of choice3

• RPMI 1640 tissue culture medium: serumfree and supplemented with 15% FBS

2 Add the appropriate volume of the lipophilic transfection reagent to each well and incubate the plate at room temperature for 30 min."

3 During this incubation, prepare serum-free medium containing plasmid DNA at a final concentration of 15 ng/mi

4 Add 0,625 ml of this mixture to each well of the 6-well plate at the completion of the incubation.

5 Incubate the plate for an additional 15 min at room temperature.

6 During the 15 min incubation, harvest the recipient macrophages as appropriate (see Chapters 1 and 2).

7 Wash the macrophages once with serum-free RPMI1640 by centrifugation at 300 g, 10 min, 4°C.

8 Resuspend the cells at 6 x 106 to 2 x 107 cells/ml in serum-free medium.

9 Add 250 jd of this cell suspension to the appropriate wells of the 6-well plate.c

11 Add 3 ml of pre-warmed RPMI 1640 supplemented with 15% FBS to each well in the plate and return cells to the incubator,

12 Incubate the transfected macrophages for 24 h prior to further stimulation and/or assessment of reporter gene expression.

a A number of effective lipophilic transfection reagents are now commercially available. Several of these reagents should be tested to determine the best reagent for the macrophage population under study.

h The optimal amount of the lipophilic transfection reagent to be used will vary based on the macrophage population and must be determined empirically.

The number of cells required for transfection will vary from population to population, but will likely be within the given range.

Protocol 7

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