To test the role of genes identified in th C. elegans screens on macrophage immune response, siRNAs (Dharmacon; pools of four siRNA duplexes per gene) were transfected into mouse macrophage cell line J774A.1 by using an Amaxa Nucleofector 96-well shuttle.
Transfections were carried out with 2 µM siRNA and 100,000 cells per well. 24 to 36 h after transfection, ultrapure E. coli was added to a final concentration of 20 ng/ml. After 5 h, supernatant was collected, and cytokine production was assayed. Cell viability was monitored, and cell number was normalized by using fluorescin diacetate; cytokine production was normalized relative to a negative control siRNA.
siRNAs were initially tested in triplicate; siRNAs that prevented IL-6 production were then assayed at least three more times.