Gene Expression in HEK293 Suspensions

This is intended as a guide only; for full experimental details please read the references provided.

Transient Gene Expression in HEK293 Suspensions using PEI STAR™ Transfection Reagent


  • PEI STAR™, 1 mg/mL, pH neutral, sterile-filtered
  • HEK293 expression medium maintained at 37°C

Transfection is inhibited by serum. Use media that is reduced-serum, serum-free ("SFM") or chemically defined ("CDM"). Here are some popular media:

Vendor Suitable Media
Thermo Fisher Scientific

Gibco™ Opti-MEM™ I Reduced Serum Media

Gibco™ FreeStyle™ 293 Expression Medium

Gibco™ Expi293™ Expression Medium (with Expi293F™ Cells)


HyClone HyCell TransFX-H

HyClone SFM4Transfx-293 media

FUJIFILM Irvine Scientific   BalanCD HEK293 (Recommended)
  • HEK293 cell culture at viable cell density of 1.5 to 2.0 x 106 cells/mL
  • Transfection-grade plasmid DNA (pDNA) with gene of interest, 1 μg per mL of culture to be transfected
  • Optional Positive Control: GFP-encoding pDNA


General Guidance

Cell densities used are for typical HEK293 cultures, with maximum viable cell densities of ~4.0 x 106 cells/mL. If using a high-density system, increase the values for cell density linearly. For example, Expi293™ media and Expi293F™ cells can support viable cell densities over 12 x 106 cells/mL instead of ~4.0 x 106 cells/mL, and so should be transfected at 3.15 x 106 cells/mL instead of 1.05 x 106 cells/mL.

The typical pDNA and PEI concentrations (1.0 and 3.0 mg/L, respectively) can achieve high transfection efficiency at viable cell densities from 1.0 x 106 cells/mL up to 5.0 x 106 cells/mL.

If performing the same expression many times, or many similar expressions, we would recommend co-varying on these parameters over corresponding ranges to find the optimal conditions:

Parameter Range
PEI Concentration 1.50 to 4.50 mg/(L final culture)
DNA Concentration 0.75 to 1.50 mg/(L final culture)
PEI/DNA Complex Time   5.0 to 15.0 minutes

Improvements in transfection efficiency are possible with changes outside the scope of this protocol. For further guidance on obtaining better yields please contact us at


Before Transfection

Subculture and expand cells to obtain culture with viability greater than 95% and viable cell density between 1.5 to 2.0 x 106 cells/mL at time of transfection.



  1. Immediately prior to transfection, ensure that viability is greater than 95% and viable cell density is 1.5 to 2.0 x 106 cells/mL.
  2. Dilute the viable cell density to 1.05 x 106 cells per mL.
  3. Invert pDNA and PEI STAR™ 1 mg/mL reagent containers several times to mix well.
  4. The final transfection concentration is 1 μg pDNA for each mL of culture to be transfected. First prepare 20 μg/mL of pDNA using 5% final culture volume in a clean vial. For example, 100 mL cell culture requires 100 μg pDNA, prepare 100 μg pDNA in 5 mL of fresh media.
  5. To a clean vial, transfer 3 μL PEI STAR™ 1 mg/mL for each mL of culture to be transfected. Use media to dilute to 60 μg/mL (5% final culture volume).
  6. Mix together diluted pDNA and PEI STAR™. Invert several times and allow to rest capped at room temperature for 10 minutes. Gently invert the closed container once immediately before use.
  7. Use 10 mL of pDNA/PEI mixture for each 90 mL of culture to be transfected. Gradually add mixture to culture while mixing.
  8. Incubate cells per typical conditions.


Post Transfection

If using a feed, booster, supplement, or enhancer, these can be added any time six hours post-transfection. Subcultures can also be prepared after six hours.

If using a GFP control, transfection efficiencies over 70% should be observed after 48 hours. For optimized procedures, efficiencies over 80% are reasonable.

Monitor expression levels and harvest upon observing plateaued titers. For typical processes, secreted proteins will be highest at 5 to 7 days post transfection.



Boussif et al (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc.Natl.Acad.Sci.U.S.A. 92 7297. PMID: 7638184.

Longo et al (2013) Transient mammalian cell transfection with polyethylenimine (PEI). Methods Enzymol. 529 227. PMID: 24011049.



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Gibco, Opti-Mem, FreeStyle, ExpiCHO, and ExpiCHO-S are trademarks or registered trademarks of Thermo Fisher Scientific. BalanCD is a FUJIFILM Irvine Scientific brand. HyClone, HyCell, TransFX-C are brands of Cytiva.