Introduction
The delivery of foreign DNA into eukaryotic cells is one of the most common molecular biology techniques to study biological mechanisms. However, unlike transformed cell lines, the efficient transfection of primary cells can be a difficult. EndoFectagen^® is a cationic polymer-based transfection system specifically designed and optimized for efficient transfection of primary endothelial cells. Transfection with EndoFectagen^® can be carried out in the presence of antibiotics and serum. Instead of normal two-day transfection, an optimized one-day transfection procedure can be performed for time-saving and highly reproducible transfection. One ml of EndoFectagen^® reagent is sufficient for up to 250 transfections per well in 96-well plate.

Storage/Handling
Upon receipt, aliquot and store EndoFectagen^® reagent A at -20°C, avoid repeated freezing/thawing cycles. Once thawed, store EndoFectagen^® reagent A at 4°C and use within a month. EndoFectagen^® reagent B can be kept at 4°C.

Quality Control
Each lot of EndoFectagen^® is performance tested by transfecting HUVECs (human umbilical vein endothelial cells) with Promega rhoSV-bata-Galactosidase control vector. HUVECs (Cat. No. 8000, ScienCell) are cultured with Endothelial Culture Medium (Cat. No. 1001, ScienCell). Gene expression is assayed by X-gal staining 24 hours post transfection. Typically, 30-60% transfection efficiency can be achieved (Figure 1).

Procedures for Transfecting Adherent Cells in 96-well Plate*

A. Preparation of cells
1. On the day of transfection, coat 96-well plate with poly-l-lysine at 2 µg/cm2. Incubate at 37°C for 2-24 hours. Rinse the poly-l-lysine coated wells with sterile deionized H2O twice before seeding of cells. The pre-coating of poly-l-lysine promotes endothelial cell evenly attach in 96-well plate.
2. Select a flask of endothelial cells with 60-80% confluency, harvest and dilute cells in Endothelial Culture Medium to give a final concentration of ~1.1×10⁵ cells/ml.

B. Transfection complex formation
1. Prepare plasmid DNA in sterile deionized H2O to give a final concentration of 1 µg/µl. To achieve successful transfection, high quality DNA with OD260/OD 280 of 1.8 or greater is recommended.
2. For each well, add 0.5 µl plasmid DNA, 10.5 µl sterile deionized H2O and 5 µl EndoFectagen^® reagent B into a 1.5 ml sterile plastic tube. Vortex gently and spin down briefly. Then add 4 µl EndoFectagen^® reagent A to make the total volume of the transfection mixture to be 20 µl, vortex for 5 seconds and spin down. Incubate at room temperature for 20-30 min.

C. Incubation of cells with transfection mixture
1. Plate 180 µl of cell suspension (~1.1×10⁵ cells/ml) in each well to give ~2×10⁴ cells per well.
2. Add 20 µl of transfection mixture to each well. Mix by gently rocking the plate side-to-side.
3. Culture the cells for ~ 24 hours under standard conditions, or perform a medium change after 4-6 hours incubation with transfection mixtures, replace with 200 µl fresh culture medium, and culture for additional 16-18 hours. Generally longer incubation time with transfection mixture results in increased transfection efficiency and decreased cell viability.
4. Harvest cells 24 hours post transfection and assay for gene expression.

* The amounts of cells and various transfection reagents mentioned in the instruction are recommended for performing transfection in 96-well plate. For transfection in larger size wells, the amounts of endothelial cells and transfection reagents (DNA, sterile deionized H2O and EndoFectagen^® reagents A&B) should be scaled up according to the surface area of the wells (Table 1).

Table 1. Recommended quantities of endothelial cells and EndoFectagen^® reagents per well.

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