Enhanced Viral Transduction of Mammalian Cells with Protamine Sulfate

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

Step 1: Prepare Viral Particles

Prepare viral particles (e.g., lentivirus, retrovirus, or AAV) using a standard virus production protocol.

• Concentrate the viral supernatant if necessary using ultracentrifugation or a commercial viral concentration kit.
• Titrate the virus using a functional assay (e.g., flow cytometry or qPCR) to determine the appropriate MOI (multiplicity of infection).

Step 2: Prepare Target Cells

• Culture target cells (e.g., HEK293T, primary cells, or other mammalian cell lines) in their respective growth media.
• Maintain cells in standard incubator conditions (37°C, 5% CO₂, 21% O₂).
• Plate cells at the appropriate density to achieve 30–50% confluency at the time of transduction.

Step 3: Prepare Transduction Media

• Immediately before transduction, replace the culture media with fresh DMEM or appropriate serum-free medium.
• Prepare the transduction mixture, including:
  • Viral particles (MOI determined by titration)
  • Protamine sulfate working solution (see Table 1 for stock preparation)
  • Culture medium (serum-free or low-serum)
• Recommended protamine sulfate concentration:
  • Typical range: 5–10 µg/mL
  • Optimize for specific cell types to balance infection efficiency and cell viability

Step 4: Transduction

• Gently mix the transduction media and add dropwise to plated cells.
• Centrifugation enhancement (optional): If working with hard-to-transduce cells, spin plates at 800 × g for 30–60 min at room temperature to enhance viral uptake.
• Incubate cells in transduction media for 6–24 hours under the following conditions: 37°C, 5% CO₂, 21% O₂
• After incubation, replace the transduction media with fresh growth media to remove excess viral particles.

Step 5: Post-Transduction and Cell Maintenance

• Continue culturing cells for 48–72 hours before assessing transduction efficiency.
• Selection (if applicable): If using a selectable marker (e.g., antibiotic resistance or fluorescence), apply selection after 72 hours post-transduction.