Silencer® Select Negative Control No 1 siRNA

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Add diluted siRNA to diluted siRNA negative control (1:1 ratio). Incubate for 5 minutes at room temperature and add to cells. Incubate cells for 1–3 days at 37°C.

Protocol tips
Add diluted siRNA to diluted siRNA negative control (1:1 ratio). Incubate for 5 minutes at room temperature and add to cells. Incubate cells for 1–3 days at 37°C.

Publication protocol

mASCs for transfection were cultured in CCM, harvested, and washed twice with PBS. The cells were then resuspended in resuspension buffer R (1 × 107 cells per mL; Neon Transfection System, Invitrogen, Carlsbad, CA, http://www.invitrogen.com) with 600 nM Silencer Select Pre-Designed & Validated small interfering RNA (siRNA) for mouse IL-6 (s68290; Ambion, Life Technologies, Carlsbad, CA, http://www.lifetech.com) or Silencer Select Negative Control No. 1 siRNA (NC siRNA, Ambion, Life Technologies). The final siRNA working concentration was 5 nM. For each sample, 1 × 106 mASCs were transfected in a total cell suspension of 100 μL using the Neon Transfection System (Invitrogen) using two pulses (1,400 V input pulse voltage/20 ms input pulse width). Transfected mASCs were immediately plated on 10-cm tissue culture dishes in 12 mL of antibiotic-free CCM. The transfected cells were cultured for 30 hours, harvested, washed, and then delivered to LPS-challenged mice.

To determine the knockdown efficiency, mASCs transfected with IL-6 siRNA were plated in six-well plates in antibiotic-antimycotic-free CCM for 30 hours, and the medium was then changed to CCM with antibiotics and antimycotics followed by addition of LPS (30 ng/mL) to activate the cells. Twenty hours later, the cells were harvested for RNA extraction using RNeasy mini kit (Qiagen, Valencia, CA, http://www1.qiagen.com), and real-time reverse transcriptase polymerase chain reaction (RT-PCR) was conducted to quantify the knockdown efficiency. Untransfected cells in the presence or absence of LPS were used as controls.

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