Silencer® Select_Vamp7 siRNA(r)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Amaxa nucleofection: RBL‐2H3 cells were resuspended at 1e6 cells/100 μl in Amaxa nucleofection solution L (Amaxa) [26] and transferred to electroporation cuvettes, and siRNA was added at various concentrations, and cells were electroporated using Amaxa program L‐029. Five hundred microlitres of prewarmed culture media was added to each cuvette, and cells were then added to 1.5‐ml prewarmed culture media in 6‐well plates. Cells were incubated for 24–48 h, and cells were assessed for transfection efficiency [using 100 nm siGLO siRNA or 2 μg of a green fluorescent protein (GFP)‐expressing plasmid; pmaxGFP (Amaxa)] by fluorescence microscopy or by using flow‐cytometry on FACScan (BD Biosciences, Franklin Lakes, NJ, USA) using a FITC filter. For siRNA knock‐down experiments, cells were incubated for 24–48 h and then washed and lysed for RNA isolation or SDS‐PAGE/immunoblotting or transferred to 24‐well plates for degranulation assays.

Protocol tips

Amaxa nucleofection: RBL‐2H3 cells were resuspended at 1e6 cells/100 μl in Amaxa nucleofection solution L (Amaxa) [26] and transferred to electroporation cuvettes, and siRNA was added at various concentrations, and cells were electroporated using Amaxa program L‐029. Five hundred microlitres of prewarmed culture media was added to each cuvette, and cells were then added to 1.5‐ml prewarmed culture media in 6‐well plates. Cells were incubated for 24–48 h, and cells were assessed for transfection efficiency [using 100 nm siGLO siRNA or 2 μg of a green fluorescent protein (GFP)‐expressing plasmid; pmaxGFP (Amaxa)] by fluorescence microscopy or by using flow‐cytometry on FACScan (BD Biosciences, Franklin Lakes, NJ, USA) using a FITC filter. For siRNA knock‐down experiments, cells were incubated for 24–48 h and then washed and lysed for RNA isolation or SDS‐PAGE/immunoblotting or transferred to 24‐well plates for degranulation assays.

Publication protocol

Amaxa nucleofection: RBL‐2H3 cells were resuspended at 1e6 cells/100 μl in Amaxa nucleofection solution L (Amaxa) [26] and transferred to electroporation cuvettes, and siRNA was added at various concentrations, and cells were electroporated using Amaxa program L‐029. Five hundred microlitres of prewarmed culture media was added to each cuvette, and cells were then added to 1.5‐ml prewarmed culture media in 6‐well plates. Cells were incubated for 24–48 h, and cells were assessed for transfection efficiency [using 100 nm siGLO siRNA or 2 μg of a green fluorescent protein (GFP)‐expressing plasmid; pmaxGFP (Amaxa)] by fluorescence microscopy or by using flow‐cytometry on FACScan (BD Biosciences, Franklin Lakes, NJ, USA) using a FITC filter. For siRNA knock‐down experiments, cells were incubated for 24–48 h and then washed and lysed for RNA isolation or SDS‐PAGE/immunoblotting or transferred to 24‐well plates for degranulation assays.

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Manufacturer protocol

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