The protocol for transfection of mammalian cells
Store at 4ºC
Introduction to NEOFECTAMINE 3000 Transfection Reagent
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Product Overview |
NEOFECTAMINE 3000 Transfection Reagent is a strong transfection reagent that ensures effective and reproducible transfection with invisible toxicity. It is formulated by unique chemistry (covalently crosslinking cationic lipids with polymer), giving rise to exceptional transfection efficiency with distinguishable features in comparison of other types of reagents. NEOFECTAMINE 3000 Transfection Reagent was shown to deliver genes to various established cell lines as well as primary cells including HEK293, 293T, 293E, CHO, COS1, HeLa, NIH 3T3, insect cell lines (Sf9 and Sf21) and a variety of other eukaryotic cell lines. NEOFECTAMINE 3000 Transfection Reagent, 1.0 ml, is sufficient for 300 to 600 transfections in 24 well plates or 50 to 100 transfections in 6 well plates.
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Application |
- Deliver DNA/RNA into mammalian, insect cells
- Enhance (up to 200 times higher efficiency) virus infection of hard
- totransfect cells,
e.g., replication-deficient Adenovirus
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Features |
- Low cytotoxicity for most of tumor cell lines and primary cells
- High transfection efficiency for a broad range of cell types
- Efficient transfection with or without serum - Simple and robust transfection procedure
- Effectively transfects both adherent and suspension cell cultures - Exceptional high titers of virus production
- Excellent for long DNAs (up to 25 kb), for single or multiple DNA
transfections, and for hard-to-transfect cells
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Storage Condition |
Store at 4 ºC. If stored properly, the product is stable for 12 months or longer.
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Broad Transfection Spectrum for Mammalian Cell Types |
| Cell Lines |
Transfection Efficiency (% GFP) |
Cell Lines |
Transfection Efficiency (% GFP) |
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| 3LL |
51% |
K562 |
22% |
| B16-F10 |
72% |
L929 |
52% |
| BAEC |
52% |
MCF-7 |
51% |
| BHK-21 |
86% |
MDCK |
52% |
| Ca Ski |
78% |
Neuro 2A |
72% |
| CaCo2 |
32% |
NIH 3T3 |
88% |
| CHO |
83% |
PC12 |
34% |
| HCS-2/8 |
42% |
SH-SY5Y |
14% |
| HEK-293 |
80-90% |
SiHa |
62% |
| HeLa |
81% |
SKOV3 |
52% |
| HLMEC |
50-65% |
HUVEC |
51% |
| H-MVEC |
49% |
IGROV1 |
21% |
| Huh-7D12 |
22% |
Jurkat |
5%-15% |
| ATT20 |
38% |
6CSFMEo |
73% |
| SK-N-SH |
20% |
WEHI 231 |
25% |
| McArdle 7777 |
60% |
SAOS-2 |
52% |
| Hep3D |
78% |
SN56 |
69% |
| SHEP |
71% |
MC3T3-E1 |
80% |
| MDA-MB-231 |
38% |
BT474 |
46% |
| 3T3 -442A |
10% |
C 2C 12 |
46% |
| COS-7 |
60-70% |
Primary mouse keratinocytes |
40-50% |
| CV-1 |
53% |
Primary human pre-adipocytes |
40-50% |
| D 407 |
62% |
Primary human
skin fibroblasts |
25% |
| DHD Pro.b |
51% |
Primary mouse embryonic fibroblast |
20% |
| LS180 |
46% |
Primary melanocyte |
46% |
| A549 |
78% |
Hela-S3 |
78% |
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Procedures for Transfecting Mammalian Cells:
1. For Adherent Cells
Cell Seeding (see Table 1): |
Cells should be plated 18 to 24 hours prior to transfection so that the monolayer cell density reaches to the optimal 80~90% confluency at the time of transfection. Serum-free DMEM medium or serum-containing medium (less than 5% serum) without antibiotics is changed to replace complete serumcontaining culture medium 1 hour before transfection.
Note: High serum levels (>5%) have a moderate inhibitory effect on NEOFECTAMINE 3000 -mediated transfections. Maximal transfection efficiencies are observed in the absence of serum. Depending upon the cell type, the presence of serum <5% may sometimes improve the overall levels of recombinant protein expression.
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| Table 1. A guideline for seeding adherent cells prior to transfection in different culture formats. |
| Culture Dish |
Surface Area(cm2) |
Number of Cells to Seed |
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| T175 flask |
175 |
0.7-1.4 × 107 |
| T75 flask |
75 |
3.0 - 6.0 × 106 |
| 100 mm dish |
58 |
2.2 – 4.4 × 106 |
| 60 mm dish |
21 |
0.9 - 1.8 × 106 |
| 35mm dish |
9.6 |
3.5 – 7.0 × 105 |
| 6-well plate |
9.6 |
4.0 – 8.0 × 105 |
| 12-well plate |
3.5 |
1.5 – 3.0 × 105 |
| 24-well plate |
1.9 |
0.8 – 1.6 × 105 |
| 48-well plate |
1 |
4.0 – 8.0 × 104 |
| 96-well plate |
0.3 |
1.2 - 2.4 × 104 |
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Preparation of NEOFECTAMINE 3000 DNA Complex and Transfection Procedures |
For different cell types, the optimal ratio of NEOFECTAMINE 3000 (μL) : DNA (μL) varies from 1:1 to 3:1. We recommend the NEOFECTAMINE 3000 (μL) : DNA (μL) ratio of 3:1 as a starting point which usually gives satisfactory transfection efficiency with invisible cytotoxicity. To ensure the optimal size of complex particles, we recommend using serum-free DMEM with High Glucose to dilute DNA and NEOFECTAMINE 3000 reagent.
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The following protocol is given for transfection in 24-well plates, refer to Table 2 for transfection in other culture formats. The optimal transfection conditions for a majority of adherent cell lines, as well as a general starting point for optimization are given in the standard protocol described below.
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- For each well, dilute 1 μg of DNA into 50 μl of serum-free DMEM with High Glucose. Vortex gently and spin down briefly
- For each well, dilute 3 μl of NEOFECTAMINE 3000 solution into 50 μl of serum-free DMEM with High Glucose. Vortex gently and spin down briefly.
- Add the 50 μl NEOFECTAMINE 3000 solution to the 50 μl DNA solution all at once. (Important: do not mix the solutions in the reverse order !)
- Vortex- mix the solution immediately and spin down briefly to bring drops to the bottom of the tube.
- Incubate for 10 minutes at room temperature
- Add the 100 μl NEOFECTAMINE 3000 / DNA mixture dropwise onto the medium in each well and homogenize the mixture by gently swirling the plate.
- For maximal transfection efficiency, change the medium to complete serum containing medium 4~5 hours post addition of NEOFECTAMINE 3000 /DNA complex.
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2. For Suspension Cells |
The following protocol is given for transfection in 6-well plate. The protocol can be scalded up or down according to culture volume.
Cell Seeding: Suspension cells are typically seeded the day of the transfection at a density of 0.5–1.0 x 106 cells per ml of culture. For optimal transfection conditions with NEOFECTAMINE 3000 , seed the number of cells adapted to the culture vessel format according to Table 3.
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| Table 2. Recommended Amounts for Different Culture Vessel |
| Culture Dish |
Culture Volume (mL)` |
Plasmid DNA (ug) |
Diluent (mL) |
Neofectamine 3000 |
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| 6-well plate |
3 |
2-4 |
0.3 |
6-12 |
| 35 mm dish |
3 |
2-4 |
0.3 |
6-12 |
| 60 mm dish |
5 |
6-12 |
0.5 |
18-36 |
| 100 mm dish |
10 |
12-24 |
1 |
36-72 |
| T75 flask |
15 |
18-36 |
1.5 |
54-108 |
| 250-mL flask |
50 |
50-100 |
2.5 |
150-300 |
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| Table 3. Recommended number of suspension cells to seed. Culture Dish Number of Cells |
| Culture Dish |
Number of Cells |
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| 96-well plate |
2 × 104- 5 × 104 |
| 48-well plate |
5 × 104 - 1 × 105 |
| 24-well plate |
1 × 105 – 2 × 105 |
| 6-well plate |
2 × 105 - 5 × 105 |
| 35 mm dish |
5 ×105 – 2 × 106 |
| 60 mm dish |
2 × 106 – 5 × 106 |
| 100 mm dish |
5 × 106 – 1 × 107 |
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NEOFECTAMINE 3000 DNA Complex Preparation and Transfection Procedures: |
The optimal ratio of NEOFECTAMINE 3000 / DNA is of 3/1 (3 μl of NEOFECTAMINE 3000 Reagent is used per 1 μg of plasmid DNA ). We recommend using serum-free DMEM with High Glucose to dilute DNA and NEOFECTAMINE 3000 Reagent to ensure the optimal size of complex particles.
The following protocol is given for transfection in 6-well plates. |
- For each well, dilute 2 – 3 μg of DNA into 100 μl of serum-free DMEM with high glucose. Vortex gently and spin down briefly.
- For each well, dilute 6 – 9 μl of NEOFECTAMINE 3000 solution into 100 μl of serum-free DMEM with high glucose. Vortex gently and spin down briefly.
- Add the 100 μl NEOFECTAMINE 3000 solution to the 100 μl DNA solution all at once (important: do not mix the solutions in the reverse order)
- Vortex- mix the solution immediately and spin down briefly to bring drops to the bottom of the tube.
- Incubate for 10 minutes at room temperature.
- Add the 200 μl NEOFECTAMINE 3000 / DNA mixture drop- wise onto the serum containing medium in each well, homogenize the mixture by gently swirling the plate.
- Incubate at 37 ºC and 5% CO2 in a humidified atmosphere.
- Transfection experiments are usually stopped after 24 to 48 hours and gene activity assessed. Cells growing in suspension are collected by centrifugation at 800 xg and then resuspended in the desired medium or buffer.
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| Storage: NEOFECTAMINE 3000 DNA In Vitro Transfection Reagent is stable for up to 18 months at 4ºC. This item is shipped at ambient temperature. |
