University of Washington School of Medicine, Department of Microbiology     

LAI Stock Preparation

Reagents

• Dulbecco’s Phosphate Buffered Saline (BioWhittaker, #17-512F)
• RPMI w/o glutamine (Fisher Scientific, #MT15040CV)
• ΔFBS: Heat-inactivated fetal bovine serum (NovaTech, Inc, #15-167-P50)
• P/S: Penicillin/Streptomycin (CellGro #30002CI)
• L-Glutamine (Fisher Scientific, MT 25-005-CI)
• Ciprofloxacin (1 gm) (Fisher Scientific, MT 61-277-RF)
• 50 ml conical tubes (Lippincott Williams & Wilkins, #366024-016)
• Hemocytometer
• Trypan Blue
• Cryovials (Corning Inc, # 2028)
• Complete RPMI = RPMI + 10% ΔFBS + 1% P/S & L-Glutamine

Protocol

Day 0

1. Infect 150 million CEM (low passage number) at MOI 0.01 – 150 µl mock or LAI.
   *For input virus, use older stocks. Make a note of the stock number (useful if stock passage number needs to be calculated)
2. Incubate in shaking waterbath at ~10 million cells/ml for 1 hour.
3. Wash 2x with warm PBS.
4. Resuspend cells in complete RPMI with Ciprofloxacin (10 µg/ml) at 1 million cells per ml (150 ml/condition). Incubate for 3 days.

Day 3

1. Spin cultures for 5 minutes at 1200 rpm, resuspend in same volume of complete RPMI without Ciprofloxacin (1 million/ml or 150 ml/condition). Incubate for 24 hours.

Day 4

1. Spin at 2000 rpm for 10 minutes. Immediately transfer supernatant into fresh flask or tubes to avoid contamination with cell pellet.
2. Aliquot in cryovials/50 ml/15 ml conical tubes.
   * Note: Virus should not be passaged too many times, good to use old stocks to generate new ones.

For specifics on appropriate handling and waste procedures please see the online chemical SOPs or our waste and spill notebook located in room 352.

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