TLR4 siRNA (m)

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	N2a cells are a mouse neuroblastoma cell line gifted by Prof. Yu‐Xian Shen (Anhui Medical University, Hefei, Anhui, China). They were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco BRL, Gaithersburg, MD) at 37°C, under a 5% CO2 humidified atmosphere. Ten percent fetal calf serum (HyClone, Marlborough, MA) and 1% penicillin (100 U/mL)‐streptomycin (100 μg/ml) were added to the medium, and the culture medium was changed every 3 days. Sequence‐specific and negative control scrambled siRNAs for mouse TLR4 gene were both purchased from Santa Cruz Biotechnology (catalogs sc‐40261, Santa Cruz Inc). Lipofectamine 2000 was used to transfect N2a cells according to the experimental protocol recommended by the manufacturer (Invitrogen, Carlsbad, CA). Briefly, first, N2a cells were incubated at 37°C under 5% CO2 for 24 hours to reach 90% confluence of the monolayer. Then, the medium containing antibiotics was removed, and the cells were washed with phosphate‐buffered saline (PBS) three times. Next, nonantibiotic medium was added. Fourth, Lipofectamine 2000 and siRNA were diluted in 250 μl of Opti‐MEM, followed by incubation at room temperature (RT) for 5 minutes and gentle mixing for 20 minutes. Finally, the mixed solution was added onto the monolayer of N2a cells. Relative experimental assays for mRNA transcription and protein expression were performed at different time points after transfection.

Protocol tips

N2a cells are a mouse neuroblastoma cell line gifted by Prof. Yu‐Xian Shen (Anhui Medical University, Hefei, Anhui, China). They were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco BRL, Gaithersburg, MD) at 37°C, under a 5% CO2 humidified atmosphere. Ten percent fetal calf serum (HyClone, Marlborough, MA) and 1% penicillin (100 U/mL)‐streptomycin (100 μg/ml) were added to the medium, and the culture medium was changed every 3 days. Sequence‐specific and negative control scrambled siRNAs for mouse TLR4 gene were both purchased from Santa Cruz Biotechnology (catalogs sc‐40261, Santa Cruz Inc). Lipofectamine 2000 was used to transfect N2a cells according to the experimental protocol recommended by the manufacturer (Invitrogen, Carlsbad, CA). Briefly, first, N2a cells were incubated at 37°C under 5% CO2 for 24 hours to reach 90% confluence of the monolayer. Then, the medium containing antibiotics was removed, and the cells were washed with phosphate‐buffered saline (PBS) three times. Next, nonantibiotic medium was added. Fourth, Lipofectamine 2000 and siRNA were diluted in 250 μl of Opti‐MEM, followed by incubation at room temperature (RT) for 5 minutes and gentle mixing for 20 minutes. Finally, the mixed solution was added onto the monolayer of N2a cells. Relative experimental assays for mRNA transcription and protein expression were performed at different time points after transfection.

Publication protocol

N2a cells are a mouse neuroblastoma cell line gifted by Prof. Yu‐Xian Shen (Anhui Medical University, Hefei, Anhui, China). They were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco BRL, Gaithersburg, MD) at 37°C, under a 5% CO2 humidified atmosphere. Ten percent fetal calf serum (HyClone, Marlborough, MA) and 1% penicillin (100 U/mL)‐streptomycin (100 μg/ml) were added to the medium, and the culture medium was changed every 3 days. Sequence‐specific and negative control scrambled siRNAs for mouse TLR4 gene were both purchased from Santa Cruz Biotechnology (catalogs sc‐40261, Santa Cruz Inc). Lipofectamine 2000 was used to transfect N2a cells according to the experimental protocol recommended by the manufacturer (Invitrogen, Carlsbad, CA). Briefly, first, N2a cells were incubated at 37°C under 5% CO2 for 24 hours to reach 90% confluence of the monolayer. Then, the medium containing antibiotics was removed, and the cells were washed with phosphate‐buffered saline (PBS) three times. Next, nonantibiotic medium was added. Fourth, Lipofectamine 2000 and siRNA were diluted in 250 μl of Opti‐MEM, followed by incubation at room temperature (RT) for 5 minutes and gentle mixing for 20 minutes. Finally, the mixed solution was added onto the monolayer of N2a cells. Relative experimental assays for mRNA transcription and protein expression were performed at different time points after transfection.

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