Citation Details
Identification of innate immunity genes and pathways using a comparative genomics approach
| Authors |  Alper S, Laws R, Lackford B, Boyd WA, Dunlap P, Freedman JH, Schwartz DA |
| In | Proc Natl Acad Sci USA (2008) 105(19): 7016-21 |
| Cells used in publication | J774A.1 (ATCC) Blood/Immune Cells Cell Lines Species: mouse Tissue Origin: ascites |
| Substrate | siRNA |
| Applications | RNAi96-well Transfection |
| Topics | Intercellular interaction/communicationSignal transduction |
| Research Area | Immunology / Hematology |
| Relevant Products |  96-well Shuttle |
Nucleofection Experiments
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.
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