Transfectamine™ 5000 Transfection Reagent

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.

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.

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.

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.

Ordering information

Price
Catalog Number
Unit Size
Quantity

Add to cart

Additional ordering information

Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
Quotation	Request
International	See distributors
Shipping	Standard overnight for United States, inquire for international

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 notes
Thaw component at room temperature before starting the experiment.

PREPARATION OF WORKING SOLUTION

Mix 2.5 ug of DNA with 200 uL of serum-free medium.
Add 7.5 uL 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 (uL) to DNA (ug) Ratio = 3 - 5 uL to 1ug

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 ug	7.5~10 ug
Serum-free medium	200 uL	600 uL
Transfectamine™ 5000 Transfection Reagent	~7.5 uL	~22.5 uL

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 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 2. 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 3. 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 4. 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 45 citations: Citation Explorer

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 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
Authors: name="60200.enl" path="C:\Users\aatbi\Dropbox (AAT Bioquest)\Website Working Files\Product References\60200.enl">60200.enlEndNote4417Correction to: Nematollahi et al., Ternary complex of plasmid DNA with NLS-Mu-Mu protein and cationic niosome for biocompatible , undefined and efficient gene delivery: a comparative study with protamine , undefined and lipofectamineArtif Cells Nanomed BiotechnolArtif Cells Nanomed Biotechnol19924682017/12/062018Dec2169-141X (Electronic)2169-1401 (Linking)29205060engPublished ErratumEngl, undefined and Artif Cells Nanomed Biotechnol. 2018 Dec;46(8):1992. doi: 10.1080/21691401.2017.1405231. Epub 2017 Dec 5.https://www.ncbi.nlm.nih.gov/pubmed/2920506010.1080/21691401.2017.1405231</electronic-reso, undefined
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

The vector-related influences of autophagic microRNA delivery by Lipofectamine 2000 and polyethylenimine 25K on mouse embryonic fibroblast cells
Authors: Lin, C. W., Jan, M. S., Kuo, J. S.
Journal: Eur J Pharm Sci (2017): 11-21

The Utilization of RNA Silencing Technology to Mitigate the Voriconazole Resistance of Aspergillus Flavus; Lipofectamine-Based Delivery
Authors: Nami, S., Baradaran, B., Mansoori, B., Kordbacheh, P., Rezaie, S., Falahati, M., Mohamed Khosroshahi, L., Safara, M., Zaini, F.
Journal: Adv Pharm Bull (2017): 53-59