Lipofectamine® 2000 Transfection Reagent

Protocol tips

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	Do not add antibiotics to the Transfection Medium

Protocol tips
Do not add antibiotics to the Transfection Medium

Publication protocol

siRNA transfection
Human chondrocytes were seeded in 6-well plates at a density of 2.0×105 cells/well in 96-well plates at a density of 3.0 ~ 8.0 ×103 cells/well for each experiment in DMEM supplemented with 10% CS. After 1 day, small interference RNAs (8 nM) for FOXO1, FOXO3, or the combination of FOXO1 and FOXO3, or Beclin1 were transfected into cells for 24 hours using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s suggested protocols. Then, culture medium was changed to DMEM with 1% CS. To confirm gene-specific silencing effects, protein lysates were prepared at 48 hours after transfection and subjected to Western blot analysis. FOXO1, FOXO3, and a scrambled control siRNA, which has no significant homology to any mammalian gene sequences were purchased from Ambion.

Plasmid transfection
Human chondrocytes were plated in 12-well plates at a density of 2.0×105 cells/well and 96-well plates at a density of 1.0×104 cells/well 18 hours before transfection. Lipofectamine 3000 (Invitrogen) reagent was used to transfect either pcDNA3 empty vector or pcDNA3-Flag-FKHR-AAA (Addgene Plasmid 13508) or pcDNA3-Flag-FKHRL1-AAA (Addgene Plasmid 10709), as recommended by the manufacturer. Six hours after transfection, culture medium was changed to DMEM with 10% CS.

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for Lipofectamine® 2000 Transfection Reagent below.

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