SignalSilence® FoxO3a siRNA

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	siRNA concentration-50 nM Add diluted siRNA to diluted Lipofectamine® RNAiMAX Reagent (1:1 ratio). Incubate for 5 minutes at room temperature and add to cells. Incubate cells for 1 days at 37°C.

Protocol tips
siRNA concentration-50 nM Add diluted siRNA to diluted Lipofectamine® RNAiMAX Reagent (1:1 ratio). Incubate for 5 minutes at room temperature and add to cells. Incubate cells for 1 days at 37°C.

Publication protocol

Calu-3 cells or primary HBEC were transfected with small interfering RNA (siRNA) specific for FOXO1 and FOXO3 (each at 50 nM; Cell Signaling Technology) using Lipofectamine RNAiMAX (Qiagen, Germany). Controls were transfected with unspecific control siRNA that does not lead to specific degradation of any cellular message (at 100 nM; Cell Signaling Technology). For experimental procedures, siRNA-transfected Calu-3 cells were incubated overnight in DMEM/F12 medium without FBS and antibiotics and subsequently treated with bacterial stressors as indicated. Primary HBEC were incubated for 24 h with insulin (1 or 5 μg/ml) and subsequently treated with bacterial stressors as indicated. Supernatants and cell lysates were kept at −80°C. Silencing of FOXO1 and FOXO3 was assessed by quantitative RT-PCR (qRT-PCR) 24 h after transfection or at the end of experimental procedures (data not shown).

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Manufacturer protocol

Download the product protocol from Cell Signaling Technology for SignalSilence® FoxO3a siRNA below.

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