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GeneXPlus Transfection Reagent

ACS-4004

GeneXPlus Transfection Reagent is a high-performance, animal-origin free, broad spectrum reagent that provides exceptional transfection of plasmid DNA into mammalian cells. This reagent affords high levels of gene expression in a variety of cell types and is suitable for both transient and stable transfection. GeneXPlus works effectively in large scale reactions, producing high protein yields. Since the reagent is composed of animal-origin free components and is serum compatible, there is no need for any culture medium change after transfection.
GeneXPlus Transfection Reagent has been optimized for use with HEK293T/17 SF suspension cells (ATCC ACS-4500) and HEKPlus SFM (ATCC ACS-4002) to reproducibly express a wide range of proteins at high levels.
Product category
Reagents
Comments

This reagent affords high levels of gene expression in a variety of cell types and is suitable for both transient and stable transfection. GeneXPlus works effectively in large scale reactions, producing high protein yields. Since the reagent is composed of animal-origin free components and is serum compatible, there is no need for any culture medium change after transfection.

 

GeneXPlus Transfection Reagent has been optimized for use with HEK293T/17 SF suspension cells (ATCC ACS-4500) and HEKPlus SFM (ATCC ACS-4002) to reproducibly express a wide range of proteins at high levels.

Applications
Cell growth and viability
Volume
1.0 mL
Storage conditions
-20°C or colder
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Documentation

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ATCC determines the biosafety level of a material based on our risk assessment as guided by the current edition of Biosafety in Microbiological and Biomedical Laboratories (BMBL), U.S. Department of Health and Human Services. It is your responsibility to understand the hazards associated with the material per your organization’s policies and procedures as well as any other applicable regulations as enforced by your local or national agencies.

Detailed product information

General

Specific applications
Transient transfection, stable transfection, gene expression, protein production, genetic manipulation.  Suitable for transfection of Bone Marrow MSCs (PCS-500-012), Dermal Microvascular Endothelial cells (PCS-110-010), THP-1 cells (TIB-202), Raw 264.7 cells (TIB-71), and SH-SY5Y cells (CRL-2266). A complete list of cell line-specific protocols can be found here.

Handling information

Handling procedure

Guidelines for optimal plasmid DNA transfection

Reaction conditions should be optimized for each cell type to ensure successful transfections. However, the general suggestions below have been demonstrated to yield high efficiency transfections using GeneXPlus Transfection Reagent. Table 1 presents recommended starting conditions based on culture vessel size.

  1. Cell seeding and Cell density at transfection. Cells should be plated 18 to 24 hours before transfection to ensure that the cells are actively dividing and reach the appropriate cell density (generally 40–80% confluence) at the time of transfection.
  2. DNA Preparation. Plasmid DNA must be sterile and free from phenol and other contaminants.
  3. Ratio of GeneXPlus Reagent to DNA. Depending on the cell type, the optimal ratio of DNA (µg) to GeneXPlus Transfection Reagent (µL) varies from 1:1 to 1:4. A DNA (µg) to reagent (µL) ratio of 1:3 is recommended as a starting point.
  4. Complex formation conditions. Prepare GeneXPlus Transfection Reagent and DNA complexes in serum-free growth medium.
  5. Presence of antibiotics and other known inhibitors. Antibiotics can inhibit transfection complex formation and therefore should be excluded from the complex formation step. Culture medium containing polyanions such as heparin, heparin sulfate or dextran sulfate can also inhibit transfection. Medium containing these chemicals should not be used for transfection; however, the medium can be replaced with medium containing polyanions 24 hours after transfection.
  6. Post-transfection incubation time. The optimal incubation time is generally 24 to 72 hours post transfection, but will vary depending on the goal of the experiment, nature of the plasmid used, and cell doubling time. Some secreted proteins can be expressed for up to 7 days post transfection.
  7. Reaction size. For protein expression, it is critical to have adequate shaking in the well. Experiments have shown that yields in a 6-well plate can be lower than those obtained in a 10 mL reaction size (or greater) in shaker flasks, but is still representative of the selected transfection conditions.

    Table 1. Recommended starting conditions for transfections with GeneXPlus Transfection Reagent

    Culture vessel

    96-well plate

    24-well plate

    12-well plate

    6-well plate

    10-cm dish

    T75 flask

    125-mL shaker flask

    Surface area (cm2)

    0.35

    1.9

    3.8

    9.6

    59

    75

    N/A

    Complete Growth Medium (mL)

    0.092

    0.5

    1

    2.5

    15.5

    19.7

    20

    Diluent (serum-free medium) (µL)

    9

    50

    100

    250

    1500

    1900

    2000

    Amount of DNA (µg)

    0.1

    0.5

    1

    2.5

    15

    19

    20

    Transfection reagent (µL)

    0.3

    1.5

    3

    7.5

    45

    57

    60


Transient plasmid DNA transfection protocol per well of a 6-well plate

Note: Adjust volumes for GeneXPlus Transfection Reagent, DNA and complete growth medium based on the surface area of the cell culture vessel as described in Table 1.

Cell Seeding
Note: For higher transfection efficiency, it is recommended that the cells are > 80% viable at the time of transfection.

  1. Approximately 18–24 hours before transfection, plate cells in 2.5 mL complete growth medium per well in a 6-well plate. Cells should be 40–80% confluent prior to transfection.

    For adherent cells: Plate cells at a density of 2 x 105 to 6 x 105 cells/well.
    For suspension cells: Plate cells at a density of 6 x 105 to 8 x 105 cells/mL.

  2. Incubate cell cultures overnight.

Preparation of Transfection Reagent:DNA complex (prepare immediately before transfection)

  1. Warm GeneXPlus Transfection Reagent to room temperature. Vortex gently before using.
  2. Place 250 μL of serum-free complete growth medium in a sterile tube.
  3. Add 2.5 μg (2.5 μL of a 1 μg/μL stock) plasmid DNA to the medium in the tube. Mix completely by gently pipetting up and down.
  4. Add 7.5 μL GeneXPlus Transfection Reagent to the diluted DNA mixture. Do not allow reagent to come in contact with the sides or bottom of the tube. Mix completely by gently pipetting.
  5. Centrifuge briefly to collect reaction mixture in bottom of the tube.
  6. Incubate at room temperature for 15–30 minutes to allow sufficient time for complexes to form.

Addition of Complexes to Cells

  1. Add the transfection complex drop-wise, to the 6-well plates containing cells in complete growth medium (cell seeding step). Swirl plate gently after each addition.
  2. Gently rock the culture vessel back-and-forth and from side-to-side to evenly distribute the GeneXPlus Transfection Reagent:DNA complexes.
  3. Incubate for 24 to 72 hours. It is not necessary to replace the complete growth medium with fresh medium.
  4. Harvest cells and assay as required.

For generating stable cell transfectants: Passage cells 24 to 48 hours post-transfection in complete growth medium containing appropriate selection antibiotics, such as G418 or Hygromycin B. Maintain selection for 1 to 2 weeks to allow for selection of cells that have undergone stable integration of DNA.

Large-scale transfections: Table 1 recommends starting volumes for 20-mL transfections (125-mL shaker flask). The volumes listed can be directly scaled up in proportion to the culture volume, if larger volumes are desired.

Quality control specifications

Functional tests
Functional Assay

History

Depositors
ATCC

Legal disclaimers

Intended use
This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use.

This product is intended for research use only. It is not intended for any animal or human therapeutic or diagnostic use.

Warranty

The product is provided 'AS IS' and the viability of ATCC® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.  Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

Disclaimers

This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use. Any proposed commercial use is prohibited without a license from ATCC.

While ATCC uses reasonable efforts to include accurate and up-to-date information on this product sheet, ATCC makes no warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. ATCC does not warrant that such information has been confirmed to be accurate or complete and the customer bears the sole responsibility of confirming the accuracy and completeness of any such information.

This product is sent on the condition that the customer is responsible for and assumes all risk and responsibility in connection with the receipt, handling, storage, disposal, and use of the ATCC product including without limitation taking all appropriate safety and handling precautions to minimize health or environmental risk. As a condition of receiving the material, the customer agrees that any activity undertaken with the ATCC product and any progeny or modifications will be conducted in compliance with all applicable laws, regulations, and guidelines. This product is provided 'AS IS' with no representations or warranties whatsoever except as expressly set forth herein and in no event shall ATCC, its parents, subsidiaries, directors, officers, agents, employees, assigns, successors, and affiliates be liable for indirect, special, incidental, or consequential damages of any kind in connection with or arising out of the customer's use of the product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, ATCC is not liable for damages arising from the misidentification or misrepresentation of such materials.

Please see the material transfer agreement (MTA) for further details regarding the use of this product. The MTA is available at www.atcc.org.

References

Curated Citations

Yang H, et al. Effect of TAK1 on osteogenic differentiation of mesenchymal stem cells by regulating BMP-2 viaWnt/β-catenin and MAPK pathway. Organogenesis 14(1):36-45, 2018. PubMed: 29913119

Frequently Asked Questions

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