Transient transfection protocol for HEK293T cells
Protocol created by Lorena Maestre - Monoclonal Antibodies Unit, Centro Nacional de Investigaciones Oncológicas
Transfected cells are important in investigating the specificity of antibodies and also permit studies on the regulation and function of proteins. Transient gene expression is the temporary expression of genes resulting from introducing foreign genetic material into a eukaryotic cell. A variety of techniques, e.g. chemical and biological methods, can be used to transfect cells.
Plasmids containing a reporter or 'tag' gene (e.g. MYC or GST) in addition to the DNA encoding the target antigen of interest are commonly used. The high copy number of the DNA plasmid results in the high expression of the target antigen. Results can be seen after 24 to 96 hours of culture but the foreign genetic material does not integrate into the chromosomes and so the target antigen is lost during prolonged culture. siRNAs, miRNAs and mRNAs can also be used for transient transfections.
A range of different eukaryotic cell lines (e.g. HeLA, COS and HEK293T) can be used and the success rate and duration of target antigen expression depends upon the genetic material introduced and the type of eukaryotic cells used. In our experience, HEK293T cells are easily transfected and the percentage of transfection can be around 80% by using the present protocol.
- HEK293T - Human embryonic kidney cell line (293T CRL-3216, from the ATCC cell bank, USA or 293T ACC 635 from the DSMZ cell bank, Germany).
- Growing in exponential growth phase in T25 cm² or T75 cm² flasks in complete medium (cells at 70-80% confluency).
Culture medium and equipment:
- Opti-MEM (Thermo Fisher, 31985070).
- CANFAST™ Transfection Reagent (Canvax, T0083).
- Complete medium: RPMI 1640 medium (Sigma, R8758-500ML) + 10%FBS+1%Penicillin/Streptomycin.
- Water bath.
- Two x 2ml eppendorf microfuge tubes.
- A sterile environment.
- Warm the Opti-MEM in a 37°C water bath.
- Check the flasks of the HEK293T cell cultures. The cells must be between 70-80% confluent.
- Remove the complete medium from the flasks and add the volume of Opti-MEM according to the size of the flasks as shown below:
Volume of Opti-MEM according to the size of the flasks Flask size Volume of Opti-MEM T25 cm² 2 ml T75 cm² 8 ml
- Label the 2 eppendorf microfuge tubes A or B.
- Add the volumes shown in the Table below to the eppendorf tubes A or B.
Eppendorf tubes A or B according to the size of the flasks Flask size Add to eppendorf A Add to eppendorf B T25 cm² 157µl Opti-MEM + 7µg plasmid vector 157µl Opti-MEM + 20µl CANFAST T75 cm² 470µl Opti-MEM + 20µg plasmid vector 470µg Opti-MEM + 60µl CANFAST
- Add the contents of eppendorf B into eppendorf A slowly and wait for 20 minutes.
- Add the mixture to the flask containing the HEK293T cells all at once and shake gently making cross movements to ensure good mixing. You can also tap the flask to loosen the cells. The transfection is more efficient if the cells are in suspension, so you can tap the flask to loosen the cells).
- Place the flask in the CO₂ incubator for at least 6 hours.
- Add 3 ml (for the T25 cm² flask) or 10 ml (for the T75 cm² flask) of complete medium.
- Place the loosely stoppered flask in the CO₂ incubator and leave for 48 hours.
- The transfected cells are then ready to study the expression of the protein of interest using the technique of your choice.
Transfected cells expressing your target antigen are valuable tools in antibody validation, especially if you want to check antibody cross reactivity with other proteins that share high homology. The cells can then be used to make cytospins for immunocytochemical labelling or cell pellets for Western blotting studies.
Figure 1. Validation of the reactivity and specificity of the anti-CD85A mAb FRAS92B. A) Immunocytochemical staining for CD85 family members on cytocentrifuge preparations using FRAS92B mAb. Staining of antibody FRAS92B was observed in HEK-CD85A-Myc transfectants but not in HEK-CD85B, CD85C, CD85D, CD85E, CD85F, CD85G, CD85H, CD85I, CD85J and CD85K. The anti-Myc, anti-GFP and anti-V5 mAbs were used to confirm transfection efficiency. B) The specificity of the antibody was also confirmed by the Western blotting technique using the transfected cell extracts. A strong band corresponding to CD85A was detected only in the HEK-CD85A cell extract.