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Refaeli Lab Lentiviral Infection Protocol - National Jewish Health

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Lentiviral Infection Protocol

Lentiviral Infection of T, B and BM Cells

 

Media

  • C10: Lymphocytes: RPMI 1640 + 10% FCS, L-glu, pen/strep, hepes, NEAA, Na-pyruvate and 2-BME
  • D10: Lymphocytes: DMEM + 10% FCS, L-glu, pen/strep, hepes, NEAA, Na-pyruvate and 2-BME
  • Bone Marrow: DMEM + 15% FCS, L-glu, pen/strep, IL-3, IL-6, SCF

Day 1

  • Plate 0.7x106 293FT cells in a 60mm dish in 4ml of D10 media

Day 2

  • In the early evening, begin transfection
  • For each infection, place 150 Buffer EC into a siliconized 1.5 mL eppendorf tube.
  • Add 0.5ug of PI, PII and PIII packaging vectors. Do not premix packaging vectors.
  • Add 1.0ug of viral plasmid (pLL3.7, etc)
  • Gently mix
  • Add 16ul of Enhancer
  • Gently mix, wait 5'
  • Add 32ul of Effectene
  • Gently mix, wait 5'
  • Add to plated 293FT's from day 1. Gently drip into media.
  • Incubate at 37 C.

Day 3

  • In the morning, aspirate media from the 293FT's and replace with 4.5mL of D10. Be careful to not disturb 293FT cells.
  • Plate cells for infections on Day 4. (3T3's: 3x105, B/T cells: 106

Day 4

  • Prepare a 15mL conical tube for each sample
  • Pipet media from 293FT's into 15mL conicals
  • Replace media on 293FT's with 4.5mL of D10. Be careful to not disturb 293 FT cells.
  • Add 45ul of HEPES and 90ul of polybrene
  • Spin tubes for 10' at 1500 RPM
  • Aspirate media from target cells
  • Pipet media from conical tubes onto target cells
  • Spin cells at 2k for 1 hr at RT
  • Aspirate media and replace with C10 (Lymphocytes) or D10 (3T3's)

Day 5

  • Repeat Day 4

Appointments

1-800-222-5864

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