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[Bio101] Lentivirus Infection   

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Abstract

Materials and Reagents

  1. Cells
  2. Polybrene (hexadimethrine bromide) (Sigma-Aldrich, catalog number: H9268 )
  3. Puromycin
  4. Human or mouse cell line and appropriate growth media reagents required for cell-based assay
  5. Polybrene appropriated antibiotics for selection purpose

Equipment

  1. Tissue culture Incubator
  2. 6 cm tissue culture plates (Thermo Fisher Scientific)

Procedure

Note: Lentiviral infections should be optimized for each cell line and cell-based assay. For example, the following parameters should be tested before starting large-scale infections to determine the optimal conditions for a given experiment:

  1. Cell seeding density.
  2. Amount of lentivirus.
  3. Puromycin concentration.
  4. Timecourse
  1. Seed cells at appropriate density in 6 ml in 6 cm plates.
    1. Adherent cells: seed 1 day prior to infection.
    2. Suspension cells: seed day of infection in media containing polybrene.
  2. Add virus to cells:
    1. (Adherent cells): Remove growth media and add fresh media containing polybrene. Alternatively, remove a portion of the growth media and supplement with media containing polybrene. Adjust volumes and polybrene concentration to achieve the correct final polybrene concentration (8 μg/ml).
  3. Viral infection:
    1. Incubate cells overnight.
    2. Change media 24 h post-infection. Remove media and replace with 6 ml fresh growth media. If antibiotics selection is desired, use fresh growth media containing antibiotics.
      Note: Puromycin concentration should be optimized for each cell line; typical concentrations range from 2-5 μg/ml.
  4. Incubate cells, replacing growth media (with antibiotics, if desired) as needed every few days. Incubation periods are highly dependent on the post-infection assay.
    Note: All lentiviral procedures should be carried out in accordance with biosafety requirements of the host institution.
Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Aboulaich, N. (2011). Lentivirus Infection. Bio-protocol Bio101: e37. DOI: 10.21769/BioProtoc.37.
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