• 10 x phosphate-buffered saline (PBS) pH 7.4: 14.8 g Na2HP04, 4.3 g KH2P04, 72.0 g NaCl, and H20 to 1 litre
• Dimethyl sulfoxide (DMSO, Sigma Chemical Company)
• Complete tissue culture medium: RPMI 1640 (Life Technologies) containing 10% endotoxin-free fetal bovine serum (FBS, Life Technologies)
1 Plate macrophages into 60 mm tissue culture dishes at a concentration of 5 x 10c cells in a total volume of 6 ml complete medium.
Protocol 5 continued
3 At the time of transfection, prepare sufficient TBS-DD to provide 1.75 ml for each plate to be trans fected.
4 Add the appropriate amount of plasmid DNA to the TBS-DD solution, mix by inversion, and warm to 37°C.i
5 Remove the growth medium from the macrophage monolayers by aspiration.b
6 Wash each monolayer gently with 2 ml pre-warmed TBS-D by gently racking the dishes manually from side to side.
7 Aspirate the wash buffer from the dishes and replace with 1.5 ml of the plasmid-containing TBS-DD mixture per plate,
8 Incubate plates at 37 °C and 5-7% C02 in a humidified incubator for 2-4 h.[
9 Remove the transfection mixture by aspiration and add 1 ml of a PBS/10% DMSO mixture."
10 Incubate for 1 min at room temperature.
11 Remove the PBS/DMSO by aspiration, and gently wash the monolayers three times with TBS-D as in step 6.
12 Remove the wash buffer by aspiration and add 6 ml of complete medium to each plate.
13 Incubate dishes at 37 "C for 18-24 h prior to further stimulation and/or assessment of reporter gene expression.
a The optimal plasmid concentration will vary based both on the target macrophage population as well as the plasmid, and should be determined experimentally in each case. b Work with only two dishes at a time, to prevent drying and detaching of the macrophage monolayer.
c The duration of the transfection should be experimentally determined in order to maximize transfection efficiency and reporter gene expression and minimize toxicity to the target cells.
Transfection of macrophages
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