实验概要
293fectin™ is a proprietary, cationic lipid-based formulation for transfection of DNA into eukaryotic cells. 293fectin™ is optimized for transfection of suspension 293 human embryonic kidney cells (e.g. FreeStyle™ 293-F cells, Cat. no. R790-07) in defined, serum-free FreeStyle™ 293 Expression Medium, and is intended for use with the FreeStyle™ 293 Expression System (Cat. no. K9000-01).
实验步骤
1. Transfecting Suspension Cells
Follow the protocol below to transfect suspension 293 cells in a 30 ml volume. You may keep the cells in FreeStyle™ 293 Expression Medium during transfection. Do not add selection antibiotics to media during transfection, as this may decrease transfection efficiency. We recommend including a positive control and a negative control (no DNA, no 293fectin™) to help you evaluate your results.
1) The day before transfection, determine the number of cells needed for transfection. For each 30 ml transfection, you will need 3 × 107 cells in 28 ml of FreeStyle™ 293 Expression Medium. Expand cells accordingly, taking into account the cell doubling time. For FreeStyle™ 293-F cells, this equates to passing cells at ~6–7 × 105 cells/ml.
2) On the day of transfection, determine the viability and the amount of cell clumping from a small aliquot of cells using the trypan blue dye exclusion method. Vigorously vortex for 45 seconds to break up cell clumps and determine total cell counts using a Coulter Counter or a hemacytometer. Viability of cells must be over 90%. Important: For best results, make sure to have a single cell suspension. It may be necessary to vortex the cells vigorously for 10 to 30 seconds to break up cell clumps.
3) Calculate the volume of cell suspension containing the number of cells needed for one transfection (you will need 3 × 107 cells for each 30 ml transfection). Place the shaker flask containing cells in a 37°C incubator on an orbital shaker.
4) For each transfection sample, prepare lipid-DNA complexes by performing.
The following:
i. Dilute 30 μg of plasmid DNA in Opti-MEM® I to a total volume of 1 ml. Mix gently.
ii. Dilute 60 μl of 293fectin™ in Opti-MEM® I to a total volume of 1 ml. Mix gently and incubate for 5 minutes at room temperature.
Note: Longer incubation times may result in decreased activity.
iii. After the 5 minute incubation, add the diluted DNA to the diluted 293fectin™ to obtain a total volume of 2 ml. Mix gently.
iv. Incubate for 20–30 minutes at room temperature to allow the DNA- 293fectin™ complexes to form.
5) While the DNA-293fectin™ complexes are incubating, remove the cell suspension from the incubator and add the appropriate volume of cell suspension (see step 3) into a sterile, disposable 125 ml Erlenmeyer shaker flask. Add fresh, pre-warmed FreeStyle™ 293 Expression Medium to a total volume of 28 ml for each 30 ml transfection.
6) After the DNA-293fectin™ incubation is complete, add the 2 ml of DNA-293fectin™ complex (from step 4) to each shaker flask containing the cell suspension. To the negative control flask, add 2 ml of Opti-MEM® I instead of DNA-293fectin™ complex. Each flask should have a total volume of 30 ml, and contain approximately 1 × 106 viable cells per ml.
7) Incubate the cells in a 37°C incubator with a humidified atmosphere of 8% CO2 in air on an orbital shaker rotating at 125 rpm.
8) Harvest cells or media (if recombinant protein is secreted) at approximately 48 hours post-transfection and assay for recombinant protein expression.
2. Transfecting Adherent Cells
1) Follow the protocol below to transfect adherent 293 cells in 24-well plates. If you are using larger- or smaller-sized tissue culture plates, vary the transfection conditions (i.e. seeding density, amount of DNA, 293fectin™, and medium) in proportion to the difference in surface area.
2) The day before transfection, trypsinize and count the cells. Plate the cells at a density of 2 × 105 cells/well so that they are 90-95% confluent on the day of transfection. Plate cells in 0.5 ml of their normal growth medium containing serum. Do not use selection antibiotics.
i. On the day of transfection, prepare lipid-DNA complexes for each sample by performing the following:
ii. Dilute 0.8–1.0 μg of plasmid DNA in Opti-MEM® I to a total volume of 50 μl. Mix gently.
iii. Dilute 2–3 μl of 293fectin™ in Opti-MEM® I to a total volume of 50 μl. Mix gently and incubate for 5 minutes at room temperature.
Note: Longer incubation times may result in decreased activity.
iv. After the 5 minute incubation, add the diluted DNA to the diluted 293fectin™ to obtain a total volume of 100 μl. Mix gently.
v. Incubate for 20–30 minutes at room temperature to allow DNA-293fectin™ complexes to form.
3) Add 100 μl of the DNA-293fectin™ complex to each well and mix gently by rocking the plate back and forth.
4) Incubate the cells in a 37°C incubator for 24–48 hours before assaying for recombinant protein expression.
注意事项
To transfect suspension 293 cells in FreeStyle™ 293 Expression Medium, use the optimized conditions below. T perform transfection experiments in a larger volume, simply scale up the volume of reagents accordingly.
Final transfection volume: 30 ml.
Number of cells to transfect: 3 × 107 cells (final cell density: 1 × 106 cells/ml) cultured in FreeStyle™ 293 Expression Medium. Make sure that the cells are healthy and greater than 90% viable before proceeding to transfection.
Amount of plasmid DNA: 20–40 μg (we typically use 30 μg).
Amount of 293fectin™: 40–80 μl (we typically use 60 μl). Use 2 μl 293fectin™ per 1 μg of plasmid DNA transfected.
