24-well Plate Spin Infection


Posted by tchan
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Protocol

Infect Raji B cells using lentivirus by spinning in a centrifuge.

Materials: 
  • Polybrene
  • Virus
  • 24-well plate
  • Complete Media (RPMI + 2% FBS + P/S/B-mercaptoethanol)
  • Raji B Cells
Methods: 
  1. Count cells.
  2. Pre-heat the centrifuge to 33°C by spinning.
  3. For each well, plate 500,000 cells in 1 ml of complete media (RPMI, 10% FBS, p/s/β-mercaptoethanol) supplemented with 8 ug/ml polybrene. Use as many wells as necessary to infect the amount of cells you want, hence 4 wells for 2 million cells…etc.
  4. Add virus to the medium. Mix by rocking plate back and forth a couple of times.  For Raji cells: MOI of 0.3 generally gives about 20% infected cells.
  5. Spin the plates at 33°C for 2 hours @ 2250 rpm.
  6. Resuspend the cells in each well by pipetting up and down. (Note: Cells tend to stick to the edges of the plate)
  7. Transfer the cells  of each well to a 6-well plate.  Add 4 mL of complete media to each well.  Incubate overnight at 37°C.  If cells need to grow over the weekend, transfer cells to a 10-cm plate and  resuspend in 12 mL of complete media.  If more than 1 million cells are infected AND the cells need to incubate over the weekend, transfer cells to a 15cm plate and resuspend in 30 mL of complete media.

NOTE: Virus infections need to be performed in the viral hood. Wear a lab coat and gloves. All materials that come in contact with virus need to be treated with 10% bleach for approximately 1 hour before being discarded!! After contact with virus, gloves need to be removed and discarded before touching anything else!!