GFP-VLPs and flow cytometry

Transfection protocol: 

Day 1: Seeding

1. Plate 6 x 15-cm plates of HEK293T cells for transfection. Cells should be split to achieve 70-90% confluence the following day (1.5E7 cells/plate for 15-cm plate). Cells seeded in 20 mL of DMEM + 10% FBS + 1% Pen/Strep.

Day 2: Transfection

Note: The total DNA used and the ratio of PEI to DNA should be optimized for each batch of PEI to ensure >70% of cells are transfected and produce maximum VLPs.

1. Aliquot 240 μg total plasmid DNA per plate in a clean centrifuge tube. Dilute in 6mL Opti-MEM
2. Separately mix 720 μL (3:1 ratio, 720 μg) of PEI (1mg/mL) in 6 mL Opti-MEM. Add dilute PEI to dilute DNA and mix quickly by pipetting up and down 5 times. Note: Can use any transfection agent as long as you get really high transfection efficiency. Adjust transfection protocol (wait time and DNA quantity) if you use a different reagent.
3. Incubate for 20 min at room temperature 
4. Add 2mL of transfection mix drop-wise to each 15-cm plate. Gently shake the plate to mix. Note: each 15-cm plate should contain 20mL of media from Day 1.

Day 3: Change media

1. Remove the media and add 15ml of fresh DMEM+10%FBS+P/S to each plate.

Day 4: Collect/filter/concentrate

1. Collect the supernatant and filter through 0.45um syringe filter (polyether sulfone, PES). This supernatant contains dilute VLPs. VLPs can be stored frozen at -80°C for storage. Otherwise, VLPs have half-life of 1 day at 37°C. 
2. Adjust volume of sup to 90mL. Add 12.3mL of precipitation solution (50% w/v polyethylene glycol 8000, 2.2% NaCl). Final concentration will be 6% PEG. Mix thoroughly and cool for 2 hours at 4°C. Incubations longer than 3hrs will result in debris and difficult to resuspend pellet.
3. Centrifuge at 2000g for 20 minutes. Remove the majority of supernatant and spin again at 2000g for 1 minute and remove the remaining supernatant.
4. Resuspend pellet in 0.9mL of PBS. Pellet may require up to 10 minutes to resuspend.
5. Can proceed with infection on the same day or freeze and store at -80°C.
    

Infection:

1. Trypsinize and resuspend 293-ACE2/TMPRSS2 cells at a concentration of 5E5/mL.
2. Add 400uL cell suspension and 50uL concentrated VLP supernatant to each well of a 24 well plate. Equivalent to 200,000 cells/well.
3. Mix by pipetting and shaking the plate to spread out the cells. Incubate overnight between 12-24hrs for optimal signal. 
4. Trypsinize and measure GFP fluorescence by flow cytometry. Set gates based on untreated cells.