MyD88 siRNA was purchased from Invitrogen (Carlsbad, CA, USA) and transfected into podocytes according to the manufacturer’s instructions. Briefly, podocytes were seeded into 6-well plates at a density of 2×105 per well in an antibiotic-free DMEM supplemented with 10% FBS and grown for 24 h at 37°C and then serum-starved for 24 h. After siRNA transfection, the cells were washed with DMEM once and cultured with DMEM containing 10% FBS for 24 h and then with DMEM containing high D-glucose or gremlin for an additional 48 h. Thereafter, the cells were harvested for protein extraction and other experimental procedures as described below. All experiments were done in triplicate and repeated at least twice.
Myd88 sirna
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
MyD88 siRNA is a pre-designed small interfering RNA (siRNA) that targets the human myeloid differentiation primary response 88 (MyD88) gene. MyD88 is an adaptor protein involved in the Toll-like receptor (TLR) signaling pathway. This siRNA can be used to knock down the expression of the MyD88 gene in cell-based studies.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Viromer green, by BioNTech (2 mentions)
Tye fluorescent labeled transfection control sirna, by OriGene (2 mentions)
Nonspecific control sirna, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Lipofectamine, by Thermo Fisher Scientific (1 mentions)
Glutamate, by Thermo Fisher Scientific (1 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (1 mentions)
Scrambled control sirna, by Santa Cruz Biotechnology (1 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (1 mentions)
7 protocols using myd88 sirna
1
Podocyte knockdown by MyD88 siRNA
2
Silencing MyD88 to Investigate PCV2 Infection
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We designed the siRNA to target myeloid differentiation primary response gene 88 (MyD88). The MyD88 siRNA and a non-specific control siRNA were chemically synthesized by Invitrogen (China). The siRNA sequences used were as follows: MyD88, 5'-AUGCCUGAGCAUUUUGAUGTT-3' (sense) and 5'-CAUCAAAAUGCUCAGGCAUTT-3' (antisense); and non-specific control siRNA, 5'-CUGCCCCAGCGAUAUCCAGTT-3' (sense) and 5'-CUGGAUAUCGCUGGGGCAGTT-3' (antisense). PAMs were transfected with 10 nM siRNA using Lipofectamine 2000 (Invitrogen, China) for 24 h and were then inoculated with PCV2 virus after washing twice with PBS buffer. The siRNA dose was optimized in pilot experiments, and no appreciable cellular toxicity was observed.
3
MyD88 and Smad7 Knockdown Protocols
4
MyD88 and Smad7 Knockdown Protocols
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For MyD88 knock-down, silencer MyD88 siRNA (Ambion by life Technologies, Carlsbad, CA) or TYE Fluorescent-labeled transfection control siRNA (OriGene, Rockville, MD) were diluted to 0.28 μM in 150 μL RPMI complete. The standard complexation protocol was employed for Viromer GREEN (Lipocalyx, Weinbergweg, Germany), according to the manufacturer’s instructions. Briefly, 0.28 μM of MyD88 or control siRNA was added to 3 μL Viromer GREEN, and the mixture was incubated at room temperature for 15 min. For each transfection, 25nM of siRNA plus Viromer GREEN mixture (10 μL) was added to each well. MyD88 knock-down was confirmed by Western Blot, as described above. For Smad7 knockdown, phosphorothioate single-stranded oligonucleotides matching the region 107–128 (5′-GCTGCGGGGAGAAGGGGCGAC-3′) of the human Smad7 complementary DNA sequence was synthesized in the sense and antisense orientation (Eurofins Genomics, Louisville KY). On day 1 post-infection, mock- or CMV-infected cells were transfected with Smad7 antisense or sense oligonucleotides (2, 4, or 10 μg/mL) by lipofectamine in serum-free media for 4h, then media was removed and replenished with RPMI complete for 48h. Smad7 knock-down was confirmed by Western Blot, as described above.
5
Glutamate-induced Cytotoxicity in PC12 Cells
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PC12 cells were graciously provided by Prof. Ohtani-Kaneko of Toyo University Life and Science. The cells were maintained in high glucose Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS, Invitrogen), 100 units/mL penicillin, and 100 mg/mL streptomycin. Cells were maintained in humidified 5% CO2 and 95% air environment at 37 °C, as described previously. Twenty-four hours before transfection, exponentially growing cells were harvested by trypsinization and replated at a density of 1 × 105 cells/cm2 with appropriate medium. The lipofectamine 2000 (Invitrogen) mediated transfection procedure was used to introduce pEGFP-C1-TLR4 plasmid DNAs into the PC12 cells. These constructs were prepared by cloning of human TLR4 cDNA into pEGFP-C1. Transcription of the TLR4 genes in each vector is controlled by the cytomegalovirus promoter. The cells were also treated with either 50 nM TRIF siRNA (Ambion) or 50 nM Myd88 siRNA (Ambion) with lipofectamine (Invitrogen) without any other treatments.
Cell viability was determined by cell proliferation colorimetric assay kit (WST assay, BioVision, Milpitas, CA), as stated in the manufacturer’s protocol. Cells were seeded on a 96-well plate and were treated with 5 or 10 mM glutamate (Fisher Scientific, Chino, CA) and 0.5 mM BSO (Sigma), which inhibits glutamate cysteine ligase.
Cell viability was determined by cell proliferation colorimetric assay kit (WST assay, BioVision, Milpitas, CA), as stated in the manufacturer’s protocol. Cells were seeded on a 96-well plate and were treated with 5 or 10 mM glutamate (Fisher Scientific, Chino, CA) and 0.5 mM BSO (Sigma), which inhibits glutamate cysteine ligase.
6
Gingival Fibroblast Transfection and Stimulation
7
Knockdown of MYD88 in Caco-2 Cells
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MYD88 siRNA was purchased from Thermo Fisher Scientific and transfected into Caco-2 cells using the Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific), according to the manufacturer’s instructions. Briefly, Caco-2 cells were plated in a 96-well plate, with antibiotic-free DMEM supplemented with 10% FBS and 1% MEM-NEAAs, and cultured overnight. After the cells reached approximately 50% confluence, the siRNA and Lipofectamine RNAiMAX complexes were added to the culture plates. After 48 h of transfection, the cells were washed and used for the assay.
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