Transfection and Stimulation of Macrophages

Macrophages were transfected using a slightly modified protocol [19] (link). Briefly, 80 µM STAT3 siRNA (Santa Cruz) or Silencer Select Negative Control (Invitrogen) in RNAiMax+OptiMEM (Invitrogen) was added to 1×10⁶ macrophages in antibiotic-free RPMI1640 medium. Cells were incubated overnight at 37°C, rinsed the next day to remove dead cells and treated with IL-4+IL-13±IL-6 for 48 h. Cells were collected for analysis via western blot or arginase assay, or stimulated with LPS to assess nitric oxide production.

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Fernando M.R., Reyes J.L., Iannuzzi J., Leung G, & McKay D.M. (2014). The Pro-Inflammatory Cytokine, Interleukin-6, Enhances the Polarization of Alternatively Activated Macrophages. PLoS ONE, 9(4), e94188.

Publication 2014

STAT3 siRNA RNAiMax+OptiMEM

Antibiotic Arginase Assay Cells Il 13 Macrophages Nitric oxide Sirna Stat3 Western blot

Corresponding Organization :

Other organizations : University of Calgary

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Variable analysis

independent variables

STAT3 siRNA
Silencer Select Negative Control
IL-4+IL-13

dependent variables

STAT3 protein expression (measured by western blot)
Arginase activity (measured by arginase assay)
Nitric oxide production (measured after LPS stimulation)

control variables

Cell type (macrophages)
Cell culture medium (RPMI1640)
Incubation conditions (37°C overnight)

controls

Positive control: Silencer Select Negative Control
Negative control: No treatment

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