Escort™ III Transfection Reagent Protocol

Product No. L3037

Introduction

Escort III 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

 

Cell lines Primary cells
COS
HepG2
Jurkat
PC-12
NIH3T3
Cardiomyocytes (rat)
Fibroblasts (rat)
Germ cells (male rat)
Hepatocytes (rat and hamster)
Keratinocytes (human)
Myoblasts (quail)
Retinal neurons (chick)
Tracheobronchial cells (human)

Storage

2 – 8°C.  DO NOT FREEZE.

Precautions and Disclaimer

This product is for R&D use only, not for drug, household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.

 

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 cells 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 III 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 – they are ready to be used for other applications. Alternatively, you can passage the cells for use in other applications.

Procedure for transfecting Jurkat cells as a stationary culture

The following protocol is designed for good efficiency using a suspension culture of Jurkat cells plated in 3.5cm tissue culture dishes (or well of a 6 well plate).


Day One: Plate Cells and Transfect

1. Plate the cells one day prior to transfection. Spin down suspension Jurkat culture and wash with PBS.  Repeat spin down and wash. Re-suspend pellet in unsupplemented basal medium at a concentration of 6 x 106 cells/mL.  Plate 0.8 mL cell solution in each 3.5 cm dish.

2. Dilute DNA and lipid reagent for complexing according to the chart below.  Use only serum-free medium or un-supplemented basal medium for dilution. In tube A, dilute 2 µL plasmid DNA in 98 µL medium.  In tube B, dilute 2 µL lipid reagent in 98 µL medium.

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

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 5-8 hours at 37°C, and then change the medium (use regular growth medium). Allow the cells in continue incubating 24 – 72 more hours.

6. Add 4mL basal medium + 12.5% Fetal Bovine Serum (FBS) to each well and incubate an additional 72 hours.


Day Three and beyond: Analyze Cells

Transfer the culture to a 10 mL centrifuge tube and spin at 400g for 10 minutes. Wash the cell pellet in PBS twice. The cells are now ready to be used in other applications.

Materials

     
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