Fusion inhibition assay using split GFP

Split GFP cell-cell fusion assay

Day 1: seeding HEK293T cells

In 96 well plate: cells will be transfected with GFP11 and will not be detached

In 6 well plate: cells will be transfected with Spike + GFP1-10 and will be detached + added to cells in 96 well plate

-              Cell seeding in 96 well plates: 8000 cells/well, to transfect the next day. If transfecting on day 3: seed 4000 cells/well

-              Cell seeding in 6 well plates: 200 000 cells/well, to transfect the next day. If transfecting on day 3: seed 100 000 cells/well

Day 2: transfection

In 96 well plates: 

• Don’t use outer wells! Keep these filled with medium but don’t use for the experiment, because evaporation can affect the readout. That leaves 60 wells/plate to use for each experiment
• Transfect with GFP11 + empty vector
  • Each well: transfect using 25 ng GFP11 + 25 ng empty vector + 0.15 µl Fugene in 5 µl Optimem
  • Calculate for your number of wells how much DNA and Fugene you need
    • Eg for 10 wells: 250 ng GFP11 + 250 ng empty vector + 1.5 µl Fugene, add optimem to a total of 50 µl
• Add everything together and incubate for 15 minutes at room temperature
• Add 5 µl DNA-Fugene mixture to well
• Note: we have also used PEI for transfection, which generally works well for HEK cells, but in 96-well format results tend to be a bit more variable 
• Controls:
  • 4 wells GFP11 + GFP1-10
  • 4 wells GFP11 only 
  • 4 wells transfected with empty vector only to add Spike + GFP1-10 transfected cells

In 6 wells: 

• Transfect with Spike + GFP1-10 
  • 4 wells
  • Each well: 500 ng Spike + 500 ng GFP1-10 + 3 µl Fugene (or PEI) in 200 µl Optimem
  • (Add dropwise to cells!)
• Controls:
  • 1 well empty vector + GFP1-10
  • 1 well untransfected

Day 3: Adding Spike + GFP1-10 cells (from 6 well plate) to GFP11 cells (96 well plate)

Transfected cells in 6 well plates: detach cells

• Remove culture medium from cells
• Add 350 µl trypsin/well 
• Check with microscope: once cells start rounding up, add 500 µl culture medium to neutralize trypsin 
• Collect detached cells 

CB6 pre-incubation + VHH incubation: 

• Add CB6 (we used 25 µl at 20 µg/ml) to 50 µl detached cells
• Incubate for 15 min at room temperature
• Add VHH/antibody of interest (we used 25 µl at 40 µg/ml, so the final concentration is 10 µg/ml in the total volume of 100 µl) 
• Incubate for 30 min at room temperature

• Remove medium from transfected cells in 96 well plate (GFP11 cells)
• Add pre-incubated cells 

• As controls, we add together
  • GFP11 cells + GFP1-10 cells (no spike) 
  • GFP11 cells + untransfected cells
  • Untransfected + GFP1-10 (no spike)

Monitor the GFP complementation signal as a result of cell-cell fusion: this typically starts to arise 2-4h after adding the cells to each other

• GFP readout: Tecan Infinite 200 Pro + take pictures with microscope to visualize cell-cell fusion and syncytium formation