SiRNA silencing human Eph receptor B4, Id: 533

Protocol tips

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	Incubate TransIT-TKO Reagent:siRNA complexe mixture at room temperature for 15–30 minutes to allow sufficient time for complexes to form and add to cells. Incubate for 4–20 h. It is not necessary to replace the complete growth medium with fresh medium

Protocol tips
Incubate TransIT-TKO Reagent:siRNA complexe mixture at room temperature for 15–30 minutes to allow sufficient time for complexes to form and add to cells. Incubate for 4–20 h. It is not necessary to replace the complete growth medium with fresh medium

Publication protocol

For transfection, two EphB4 targeting siRNAs and a control non-specific siRNA were used. HNSCC cells were transfected in serum-free, antibiotic-free growth medium using Mirus TransIT-TKO Transfection Reagent (Madison, WI, USA), according to the manufacturer’s instructions. Short interfering RNAs (siRNA) specific for human EphB4 (ID: s243, and 533) and the non-specific control siRNA were obtained from Invitrogen (Carlsbad, CA, USA). Briefly, cells were transfected using 10 μL TransIT-TKO for a final concentration of 25–50 nM siRNA. Cells were incubated with the transfection complex for 4–20 h, medium was replaced with fresh serum-containing and antibiotic-containing growth medium and used for further analysis.

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