Description

Endothelial cell transfection kit, 250 transfections in 96-well plate.

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 FibroFectagen reagent A at -20°C, avoid repeated freezing/thawing cycles. Once thawed, store FibroFectagen reagent A at 4°C and use in a month. FibroFectagen reagent B can be kept at 4°C.

Quality Control

Each lot of FibroFectagen is performance tested by transfecting Human Pulmonary Fibroblasts (HPFs, Cat. No. 3300, ScienCellT) with Promega® ?SV-bata-Galactosidase control vector. Gene expression is assayed by X-gal staining 24 hours post transfection. Typically, 40-70% transfection efficiency can be achieved (Figure 1).

Procedures for Transfecting Adherent Cells in 96-well Plate*

1. 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 2-4 hours. Rinse the poly-l-lysine coated wells with sterile deionized H2O twice before seeding of cells. The pre-coating of poly-l-lysine ensures good and even fibroblasts adhesion.
2. Select a flask of fibroblasts with 60-80% confluency, harvest and dilute cells in Fibroblast Medium to give a final concentration of ~1.1×105 cells/ml.
2. 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, 9.5 µl sterile deionized H2O and 5 µl FibroFectagen reagent B into a 1.5 ml sterile plastic tube. Vortex gently and spin down briefly. Then add 5 µl FibroFectagen 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.
3. 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 cells and transfection reagents (DNA, sterile deionized H2O and FibroFectagen reagents A&B) should be scaled up according to the surface area of the wells (Table 1).

Table 1. Recommended quantities of fibroblasts and FibroFectagen® reagents per well.

Culture Vessel	Growth Area (cm2/well)	# of cells	1µg/µl DNA stock(µl)	Sterile DI H20(µl)	FibroFectagen® reagent B (µl)	FibroFectagen® reagent A (µl)	ECM (µl)
96-well plate	0.35	20,000	0.5	9.5	5	5	180
48-well plate	0.8	45,000	1.1	22	11.4	11.4	411
24-well plate	2.0	115,000	2.9	54	29	29	1029
12-well plate	4.0	230,000	5.7	109	57	57	2057
6-well plate	9.6	550,000	13.7	261	137	137	4937