Search Thermo Fisher Scientific
Search Thermo Fisher Scientific
Lipofectamine® LTX Reagent is a proprietary, animal-origin free formulation for the transfection of DNA into eukaryotic cells with low cytotoxicity. This reference provides a recommended procedure to transfect plasmid DNA into HUVEC Human Umbilical Vein Endothelial primary isolates (passage 0, Cambrex, East Rutherford, NJ) using Lipofectamine® LTX Reagent (Cat. No. 15338-100).
Important Guidelines for Transfection
Follow these important guidelines when transfecting DNA into HUVEC cells using Lipofectamine® LTX Reagent:
Materials Needed
Have the following reagents on hand before beginning:
To transfect HUVEC cells in different tissue culture formats, vary the amounts of Lipofectamine® LTX Reagent, DNA, cells, medium and PLUS™ Reagent used in proportion to the relative surface area, as shown in the table (amounts given on a per well basis).
Culture vessel | Surface area per well1 | Volume plating medium | Cells per well | Volume dilution medium2 | DNA | Lipofectamine® LTX Reagent | PLUS™ Reagent |
---|---|---|---|---|---|---|---|
96-well | 0.3 cm2 | 100 μl | 8 x 103 | 20 μl | 100 ng | 0.25 - 0.3 μl | 0.1 μl |
48-well | 1 cm2 | 200 μl | 2 x 104 | 40 μl | 200 ng | 0.5 - 0.6 μl | 0.2 μl |
24-well | 2 cm2 | 500 μl | 4 x 104 | 100 μl | 500 ng | 1.25 - 1.5 μl | 0.5 μl |
12-well | 4 cm2 | 1 ml | 8 x 104 | 200 μl | 1 μg | 2.5 - 3.0 μl | 1.0 μl |
6-well | 10 cm2 | 2 ml | 2 x 105 | 500 μl | 2.5 μg | 6.25 - 7.5 μl | 2.5 μl |
1 Surface areas may vary depending on the manufacturer.
2 If the volume of Lipofectamine® LTX Reagent is too small to dispense accurately, and you cannot pool dilutions, predilute Lipofectamine® LTX Reagent 10-fold in Opti-MEM® I Reduced Serum Medium, and dispense a 10-fold higher amount (should be at least 1.0 μl per well). Discard any unused diluted Lipofectamine® LTX Reagent.