Generation of SC2-VLPs - Protocol for transfection and filtering in 96well format

Transfection protocol: 

Day 1:

1. Plate HEK293T cells for transfection. Cells should be split to achieve 70-90% confluence the following day (50k cells/well in 96-well plate). Cells seeded in 0.15 mL DMEM + 10% FBS + 1% Pen/Strep.

Day 2: 

    2. Aliquot 0.1875 μg total plasmid DNA per sample in PCR strip. (Note: I typically do transfections with replicates so aliquoting 0.1875xn μg of total DNA).

    3. Separately mix 0.563 μL (3:1 ratio) of PEI (1mg/mL) in 18.75 μL Opti-MEM. Scale this mastermix by number of samples and replicates. Add dilute PEI to DNA and mix quickly by pipetting up and down 5 times. 

    4. Incubate for 20 min at room temperature 

    5. Pipette the mixture gently into wells (~19uL per well). DO NOT ADD DROPWISE! Due to the shape of the wells, dropwise addition can lead to cell detachment. Gently pipette up and down to mix and shake the plate vertically and horizontally to mix thoroughly.

Day 4:

    6. Collect 65 uL of the supernatant and add to 0.45um filter plate. This supernatant contains VLPs.

    7. Place filter plate on a new TC treated white 96well plate (clear or opaque bottom) and centrifuge at 100g for 2minutes to collect filtered supernatant. Dead volume in filter plate is 15uL so the final volume of VLPs in the white plate will be 50uL.

    8. VLPs can be stored frozen at -80C for storage. Otherwise, VLPs have half-life of 1 day at 37C. 

Infection:

    9. Trypsinize and resuspend 293-ACE2/TMPRSS2 cells at a concentration of 1E6/mL.

  10. Add 50uL cell suspension to 50uL filtered VLP supernatant TC-treated 96 well plate. Equivalent to 50,000 cells/well.

  11. Mix by pipetting and shaking the plate to spread out the cells. Incubate overnight between 12-24hrs for optimal signal. Signal decreases ~2-fold by 48hrs.

  12. Remove supernatant and add 20uL of 1x passive lysis buffer and incubate on shaker for 20 minutes at RT.

  13. Add 50uL of luciferase assay buffer to each well using a multichannel or autoinjector on the Tecan SPARK. Measure using the luminescence plate reader. Minimize the time for this step as the signal will decline quickly after addition of the buffer.