Retroviral Infection Protocol

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 Media: DMEM + 15% FCS, L-glu, pen/strep, IL-3, IL-6, SCF

 

Preparing T cells for Infection

• Plate 106 purified CD4+ T cells
• Activate with 1ug/ml of anti-CD3 and anti-CD28
• Infect after 24hrs

 

Preparing B cells for Infection

• Plate 106 purified CD4+ T cells
• Activate with 1ug/ml of anti-IgM and anti-CD40
• Infect after 24hrs

 

Preparing Bone Marrow cells for Infection

• Treat mice with 5mg/mouse of 5FU
• Collect BM cells from mice leg bones.
• Lyse red blood cells
• In a 24 well plate, plate 2x106 BM cells per well in Bone Marrow Media.
• Infect after 24hrs

 

Protocol

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 1.5mL eppendorf tube.
• Add 1.5ug of pCL-ECO packaging vector.
• Add 1.75ug of viral plasmid (pMIG)
• 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. See below for protocols for B and T cell preparation.

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) or BM supplemented D10.

Day 5

• Repeat Day 4