Lipofectamine® 2000 Transfection Reagent

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	Do not add antibiotics to the Transfection Medium

Protocol tips
Do not add antibiotics to the Transfection Medium

Publication protocol

Mfn2 Overexpression
To increase Mfn2 expression, we transfected the
PASMCs with a plasmid-carrying pEGFP Mfn2 complementary
DNA (cDNA), which was built and synthesized by GenePharma (Shanghai, China). The sequence of Mfn2 cDNA is as
follows—sense: 50
-cggaattcATGTCCCTGGCTCTTCTCTC-30
,
antisense: 50
-cggatccCTATCTGCTGGGCTGCAG-30
. Nontargeted control cDNA (shNC) was used as a negative control.
PASMCs were trypsinized, counted, seeded into 6-well plates,
and cultured in 2 mL of DMEM/F12 with 10% FBS until they
reached 30%–50% confluence. Then, the cells were incubated
in serum-free DMEM/F12 for 24 hours. For each well, we
diluted 3.2 mg of plasmid and 8 mL of Lipofectamine 2000
reagent with 245 mL of OPTI-MEM medium separately. Then,
we mixed 2 diluents and incubated mixture at room temperature
for 20 minutes. Thus, the dilution ratio of Lipofectamine to
plasmid was 2.5:1. Next, we added the mixture to the well
and incubated the cells for 6 hours at 378C in a 5% CO2 incubator and then changed the culture medium to DMEM/F12 with
10% FBS and exposed the cells to normoxia or hypoxia. We
measured the Mfn2 protein expression by Western blot analysis
to determine the efficiency and specificity of transfection.

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Manufacturer protocol

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

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