Escort™ IV Transfection Reagent Protocol

Introduction

The Escort™ IV Transfection Reagent (Product No. L3287) is a liposome suspension composed of a polycationic lipid and a neutral, non-transfecting lipid compound. Transfection using liposomes is a commonly used method for the introduction of DNA into eukaryotic cells. This technique has been used to obtain both transient and stable transfections in a wide variety of cell types (see below). The procedure is based on the formation of a complex between the plasmid DNA and the lipid reagent, which adheres to the cell surface and is taken up by the cell, presumably by endocytosis, releasing the DNA into the cytoplasm.

Cells tested for good efficiency transfection

Mammalian cell lines	Insect Cells	Primary cells
C2C12 CHOK1 COS-1 COS-7 HEK293 HeLa LNCaP NIH3T3 T-24	S2 (D. mel.) S2R+ Sf-9 (adherent) Sf-9 (suspension) T. Ni. Sf-RVN®	Aortic smooth muscle (rat) Bone marrow endothelial cells (human) Keratinocytes (human) Myoblasts (rabbit)

Procedure for transfecting adherent primary cells and cell lines

The following protocol is designed for good efficiency across a variety of cell types. For highest transfection efficiency, use the Optimization Protocol to determine the best set of conditions for your cells.

Day One: Plate Cells

Plate the cells one day prior to transfection. Use a seeding density that will provide >70% confluence about 12 hours later, generally according the chart below:

Culture Dish	Total sells to plate (per well)
24-well plate	0.5 – 2 x105
12-well plate	0.25 - 1 x106
6-well plate or 3.5-cm dish	0.5 – 2 x106
10-cm dish	1 - 2 x106

Day Two: Transfection

1. Dilute DNA and lipid reagent for complexing according to the chart below. Use only unsupplemented basal medium for dilution. In tube A, dilute the plasmid DNA in medium. In tube B, dilute the lipid reagent in medium.

	Tube A		Tube B
Culture Dish	DNA volume (µL)	Medium volume (µL)	Escort™ IV Transfection Reagent volume (µL)	Medium volume (µL)	Approximate volume of media during transfection
24-well plate	0.5	9.5	0.5	9.5	500 µL
12-well plate	1	49	1	49	1 mL
6-well plate or 3.5-cm dish	2	98	2	98	2 mL
10-cm dish	5	495	5	495	5 mL

2. Add tube A to tube B and mix gently. Allow complexes to form 15-30 minutes at room temperature.

3. Remove half the medium from each cell culture dish and discard.

4. Add complexes drop-wise to the cell cultures. Swirl the plates gently to distribute the complexes evenly over the cells.

5. Incubate the cells 5-18 hours at 37 °C, and then change the medium (use regular growth medium). Allow the cells in continue incubating 24-72 more hours.

Day Three and beyond: Analyze Cells

Collect and lyse the cells. Alternatively, passage the cells for use in other applications.