Transfectamine™ 5000 Transfection Reagent

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Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
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Physical properties

Solvent

Water

Storage, safety and handling

Intended use	Research Use Only (RUO)
Storage	Freeze (< -15 °C); Minimize light exposure

Overview SDS Protocol

Example protocol

AT A GLANCE

Protocol Summary

Prepare cells for transfection
Prepare Transfectamine™ 5000-DNA mixture
Add Transfectamine™ 5000-DNA mixture to cell culture
Culture overnight
Analyze transfection efficiency with appropriate method

Important Note

Thaw component at room temperature before starting the experiment.

PREPARATION OF WORKING SOLUTION

Mix 2.5 µg of DNA with 200 µL of serum-free medium
Add 7.5 µL of Transfectamine™ 5000 to Step 1
Mix well and incubate at room temperature for 20 minutes.

Note: Ratio of Transfectamine™ 5000 and DNA need to be optimized for different cell line, in general: Transfectamine™ 5000 Transfection Reagent (µL) to DNA (µg) Ratio = 3 - 5 µL to 1µg

Sample protocol detail for 6-well and 10 cm plate

Component	6 well plate (per well)	10 cm plate
Fresh culture medium	2 mL	6 mL
Plasmid	~2.5 µg	~7.5-10 µg
Serum-free medium	200 µL	600 µL
Transfectamine™ 5000 Transfection Reagent	~7.5 µL	~22.5 µL

SAMPLE EXPERIMENTAL PROTOCOL

Preparation of Cell Culture

Culture cells to ~ 90% confluency at time of transfection.
Replace with fresh growth medium before transfection. For example, replace with 2 mL of medium per well for 6-well plates and 6 mL of medium for 10 cm plates.

Transtection Protocol

Add Transfectamine™ 5000 -DNA mixture to culture plate and culture overnight.

Note: Recombinant protein can start to be detected as early as 16 hours post-transfection. Maximal expression level may be observed 72~96 hours post-transfection.

Images

Figure 1. Transfection efficiency comparison of Transfectamine™ 5000 versus Lipofectamine 2000 reagents for CRISPR-Cas9-GFP plasmid in HeLa cells. Each reagent was used to transfect HeLa cells in a 96-well format, and the GFP expression was analyzed after 48 and 72 hours post-transfection. Transfectamine™ 5000 transfection reagent provided higher GFP transfection efficiency compared to Lipofectamine 2000.

Figure 2. Transfection efficiency comparison in HeLa cells using Transfectamine™ 5000, Lipofectamine 2000 and Lipofectamine 3000 reagents. Each reagent was used to transfect HeLa cells in a 96-well format, and GFP expression was analyzed 24 hours post-transfection. Transfectamine™ 5000 transfection reagent provided higher GFP transfection efficiency compared to Lipofectamine 2000 and Lipofectamine 3000 reagents.

Figure 3. Transfection efficiency comparison in CHO-K1 cells. CHO-K1 cells were cultured in 6-well plate to ~90% confluency. 2.5 ug of GFP plasmid was transfected with Lipofectamin 2000, Lipofectamine 3000 and Transfectamine™ 5000. Images were taken 24 hours post transfection with fluorescent microscope through FITC channel.

Figure 4. Cell viability comparison in CHO-K1 cells. One group of CHO K1 cells were transfected with GFP plasmid using Lipofectamine 2000, Lipofectamine 3000 and Transfectamine™ 5000, the second group of CHO K1 cells were treated with same amount of transfection reagent as the first group but without plasmid. After 48 hours, cell viability of each group was measured with Cell Meter™ Colorimetric WST-8 Cell Quantification Kit (Cat. 22770). The higher absorbance at 460nm represents more viable cells.

Figure 5. Transfection efficiency comparison in HeLa cells. HeLa cells were cultured in 6-well plate to 90% confluency. 2.5 µg of GFP plasmid was transfect with Lipofectamine 2000, Lipofectamin 3000 and Transfectamine™ 5000. Signal intensity was measured by flow cytometry.

Citations

View all 47 citations: Citation Explorer

circ\_0008285 Regulates Glioma Progression via the miR-384/HMGB1 Axis
Authors: Yan, Manli and Hu, Caihong and Hu, Qi and Ma, Heran and Lei, Changjiang and Liu, Yamei and others,
Journal: International Journal of Genomics (2023)

Discovery of paralogous GnRH and corazonin signaling systems in an invertebrate chordate
Authors: Yanez-Guerra, Luis Alfonso and Zandawala, Meet
Journal: bioRxiv (2023): 2023--03

SINE Insertion in the Intron of Pig GHR May Decrease Its Expression by Acting as a Repressor
Authors: Chen Cai, and Zheng, Yao and Wang Mengli, and Murani, Eduard and D'Alessandro, Enrico and Moawad, Ali Shoaib and Wang, Xiaoyan and Wimmers, Klaus and Song, Chengyi
Journal: Animals (2021): 1871

A Litopenaeus vannamei TRIM32 gene is involved in oxidative stress response and innate immunity
Authors: Wang, Lei and Lu, Ke-Cheng and Chen, Guo-Liang and Li, Ming and Zhang, Chao-Zheng and Chen, Yi-Hong
Journal: Fish \& Shellfish Immunology (2020): 547--555

A Powerful Transfection Reagent for Building Stable GPCR Expressing Cell Lines
Authors: Kan, Shu and Liao, Jinfang and Diwu, Zhenjun
Journal: Biophysical Journal (2020): 564a--565a

Lipofectamine 2000/siRNA complexes cause endoplasmic reticulum unfolded protein response in human endothelial cells
Authors: Li, Z., Zhang, C., Wang, Z., Shen, J., Xiang, P., Chen, X., Nan, J., Lin, Y.
Journal: J Cell Physiol (2019): 21166-21181

Transfection reagent Lipofectamine triggers type I interferon signaling activation in macrophages
Authors: Guo, X., Wang, H., Li, Y., Leng, X., Huang, W., Ma, Y., Xu, T., Qi, X.
Journal: Immunol Cell Biol (2019): 92-96

Comparison between Lipofectamine RNAiMAX and GenMute transfection agents in two cellular models of human hepatoma
Authors: Berardo, C., Siciliano, V., Di Pasqua, L. G., Richelmi, P., Vairetti, M., Ferrigno, A.
Journal: Eur J Histochem (2019): ersion="1.0" encoding="UTF-8" ?>60200.enlEndN

Correction to: Nematollahi et al., Ternary complex of plasmid DNA with NLS-Mu-Mu protein and cationic niosome for biocompatible and efficient gene delivery: a comparative study with protamine and lipofectamine
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Journal: Artif Cells Nanomed Biotechnol (2018): 1992

Ternary complex of plasmid DNA with NLS-Mu-Mu protein and cationic niosome for biocompatible and efficient gene delivery: a comparative study with protamine and lipofectamine
Authors: Nematollahi, M. H., Torkzadeh-Mahanai, M., Pardakhty, A., Ebrahimi Meim and , H. A., Asadikaram, G.
Journal: Artif Cells Nanomed Biotechnol (2018): 1781-1791