General Transfection

Introduction

This protocol describes a general method for transfecting mammalian cells using linear polyethylenimine. Transfections allow for transient expression of a gene of interest in a target cell line and can be useful for short term studies of protein function. We specifically use this protocol with a subclone of HEK293T cells optimized for viral production (AAVpro or Lenti-X), but any HEK293T cell line should work. This approach can be adapted for different cell lines and different transfection reagents.

Last Upload: November 7, 2023

Workflow Timeline

Day 0: Seed HEK293T cells (or a subclone of HEK293T optimized for viral production)

Day 1 (pm): Transfect Cells

Day 2 (am): 18 h post transfection - Remove media, replace with fresh media

Day 3 or more (am): Observe fluorescence, harvest cells, or perform your experiment

Equipment

• Class II, Type A2 Biological Safety Cabinet
• 0.5–10 µL single channel pipette
• 2–20 µL single channel pipette
• 20–200 µL single channel pipette
• 200–1000 µL single channel pipette

• Ice bucket
• CO₂ incubator
• Pipet controller
• Hazardous waste container

Reagents

• DMEM high glucose, Corning 10-013-CV/li>
• L-alanyl-L-glutamine (or alternative stable glutamine such as glutaGRO, Corning 25-015-CI)
• Heat-inactivated FBS
• Low serum medium such as Opti-MEM or Opti-Pro SFM, Thermo Fisher, 12309019
• 25 mM chloroquine

• Polyethylenimine, linear MW 25,000 Da
• Microcentrifuge tubes, Neptune 3745.X
• 10 cm tissue culture dish, Corning 430167
• Hydrochloric acid
• Sodium hydroxide
• 0.22 μm polyethersulfone (PES) filter
• Syringes for filtering

Reagent Preparation

1. DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or stable alternative such as glutaGRO)
   • To a 500 mL bottle of DMEM high glucose, add 55 mL of heat-inactivated FBS and 5 mL of 100X glutaGRO. Store at 4 °C.
   

   *Pro-Tips* Different brands and lots of FBS can promote or inhibit transfection. Test a variety of brands and lots of FBS to find one suitable with your protocols. FBS can be purchased already head inactivated or it can be inactivated in the lab by heating to 56 °C for 30 min.

2. 25 mM chloroquine diphosphate
   • Dissolve 0.129 g of chloroquine diphosphate salt into 10 mL of sterile water.
   • Filter sterilize through a 0.22 µm filter.
   • Aliquot 50–100 µL and store at -20 °C.
   • Aliquots can be thawed and stored at 4 °C prior to use. Thawed aliquots should be discarded after 1–2 months.
3. 1 mg/mL polyethylenimine, linear MW 25,000 Da (PEI)
   • Dissolve 100 mg of powder into 100 mL of deionized water.
   • While stirring, slowly add hydrochloric acid until the solution clears.
   • Check the pH of the solution
   • Use hydrochloric acid or sodium hydroxide to adjust the pH to 7.0. Typically, the solution will be basic and will need adjustment with hydrochloric acid first.
   

   *Pro-Tip* The pH of this solution will drift pretty rapidly upon addition of acid or base. Add only a few drops at a time, allow them to mix and recheck the pH to prevent over or undershooting the desired pH.
   • Allow the solution to mix for 10 min and then recheck the pH to ensure that it has not drifted.
   • Filter the solution through a 0.22 µm membrane.
   • Aliquot 500–1000 µL into sterile tubes.
   • Store the tubes at -80 °C.
     • After thawing the solution can be stored at 4 °C for up to 2 months. After 2 months, discard the tube and thaw a new working stock.

Considerations Before You Start

• The health of the cell line is critical for obtaining high levels of virus.
• HEK293T cells should be split 3 times a week:
  • Monday: Plate 1x10⁶ cells in a 75 cm² flask in a volume of 15 mL.
  • Wednesday: Plate 1x10⁶ cells in a T75 flask in a volume of 15 mL.
  • Friday: Plate 8x10⁵ cells in a T75 flask in a volume of 15 mL.
• Do not add antibiotics to the media.
• Use cells that are below passage 20 for viral production.
• The optimal mass DNA:mass PEI ratio will need to be empirically determined for each new batch of 1 mg/mL PEI prepared.
  • There may be variation between batches of PEI depending on the user, quantities of chemical used, volumes, pH adjustment etc. Consequently, each batch needs to be validated and the best ratio of mass DNA:mass PEI determined.

Procedure

1. Seed HEK293T cells at 3.8x10⁶ cells per plate in DMEM complete in 10 cm tissue culture plates.
2. Incubate the cells at 37 °C, 5% CO₂ for ~20 h.
3. Gently aspirate media, add 10 mL fresh DMEM complete containing 25 µM chloroquine diphosphate and incubate ~5 h
   • For 10 mL of DMEM complete, add 10 µL of 25 mM chloroquine diphosphate.

4. Dilute 18.9 µg of DNA into 500 µL of Opti-Pro SFM.
   

   *Pro-Tip* Endotoxins can inhibit transfection; therefore, plasmid DNA purification should include an endotoxin removal step. For high quality plasmid DNA, the plasmid should also be propagated in an endonuclease negative E. coli strain such as NEB stable.
5. Dilute 1:3 (µg DNA:µg PEI) in 500 µL total of OptiPro SFM (per 10 cm plate).
   • 56.7 µL of 1 mg/mL PEI, MW 25,000 Da in 443.3 µL of OptiPro SFM per 10 cm plate
   

   *Pro-Tip* The ratio of µg DNA:µg PEI needs to be empirically determined. Once a batch of PEI is prepared, transfect cells with a fluorescent plasmid using a variety of ratios. Check the cells 1–2 days after transfection to determine what ratio gives the highest percentage of GFP positive cells.
   • Refer to the table below for a possible range of ratios to test:

   Ratio of DNA:PEI	Amount of DNA (μg)	Volume of 1 mg/mL PEI (μL)
   1:1	18.9	18.9
   1:2	18.9	37.8
   1:3	18.9	56.7
   1:4	18.9	75.6
   1:5	18.9	94.5
   1:6	18.9	113.4

6. Gently add the diluted PEI to the diluted DNA. Add the diluted PEI dropwise while gently flicking the diluted DNA tube. Incubate the mixture 15–20 min at RT.
7. Carefully transfer the transfection mix to your HEK293T cells. Add the transfection mix dropwise, being careful not to dislodge the cells.
8. Incubate the cells for 18 h, or until the following morning.
9. The following morning, carefully aspirate the media. Replace the media with 15 mL of DMEM complete.
10. Incubate the cells 24–48 h before checking for protein expression.

Sample Data

Legend: Lenti-X 293T cells were transfected using 1:1, 1:2, 1:3 and 1:6 µg of pRosetta:µg of PEI. The 1:2 and 1:3 ratios provided high transfection efficiencies as can be seen here by the amount of green fluorescent protein expression (green in the right panels) with a limited effect on cell growth.