Lentivirus concentration

Virology Retrovirus (HIV)
1

Does anyone have a good protocol for concentrating recombinant VSV-G pseudo typed lenti particles? Thanks

2

Navneet

Go for Ultracentrifugation Protocol (Gold Standard)

Materials:

  • Lentiviral supernatant (usually 0.45 µm filtered)
  • Ultra-clear ultracentrifuge tubes
  • SW-28 or SW-32 rotor
  • 20% sucrose cushion in PBS (optional, helps with cleaner pellet)

Steps:

  1. Filter viral supernatant using 0.45 µm PES filters to remove cell debris.
  2. Layer 20% sucrose cushion (~4 mL) at the bottom of ultracentrifuge tube (optional but recommended).
  3. Gently add your viral supernatant on top (~25–30 mL per tube).
  4. Spin at 100,000 x g for 2 hours at 4°C.
  5. Carefully remove the supernatant without disturbing the pellet.
  6. Resuspend pellet in 50–200 µL cold sterile PBS, DMEM, or viral resuspension buffer.
  7. Aliquot and store at –80°C. Avoid multiple freeze-thaws.

Alternatively, if you dont have access to Ultra Centrifuge, use PEG 8000 instead.

1 Like
3

Argerine

Can you provide details on the PEG based protocol. We don’t have access to Ultra centrifuge in our lab.

4

Hi Navneet Step 1 remains the same. Filter our the supernatant using 0.45 µm PES filters to remove cell debris

  • Mix filtered supernatant with PEG-it™ Virus Precipitation Solution (or DIY: 8% PEG 8000 + 0.5 M NaCl).
  • Incubate overnight at 4°C.
  • Spin at 1,500–3,000 x g for 30 min.
  • Resuspend viral pellet in PBS or DMEM.

Dont forget to Titer after concentration to measure viral yield (e.g., via p24 assay or transduction assay). If you are working with any specific Cell types, you have to modify the protocol accordingly.