NanoBiT Luciferase-based live-cell assay for cell–cell fusion quantification

The chicken DF1 cell line was cultivated in DMEM:F12 culture medium supplemented with 4% calf serum, 4% fetal calf serum and 1% chicken serum. A fresh mixture of antibiotics was added prior to the use of the culture medium. Cells were cultivated at 37°C in a 5% CO₂ atmosphere.

Plasmid DNA used in the protocol: 
CMV HaloTag®-LgBiT (Promega), CMV HaloTag®-HiBiT (Promega), pVSV-G (Clontech), pcGag-Pol (Plachý et al. 2010), pFuTraP (Štafl et al. 2021)

A. Preparation of chicken DF1 cell lines stably expressing HiBiT or LgBiT luciferase fragments

1. After the DF1 cells reach confluency between 80%–90%, discard the culture medium, wash cells with PBS, and add prewarmed (37°C) trypsin-EDTA solution. Incubate the cells at 37°C for 5–8 min.
2. Resuspend the cells in the culture medium and determine the cell concentration.
3. For each transfection seed 4.5 × 10⁵ of DF1 cells on a 6-well plate. Cultivate the cells for 18–24 h.
4. Transfect the DF1 cells with linearized vectors (restriction digestion with AgeI-HF in CutSmart® buffer followed by inactivation of the restriction enzyme following the NEB protocol) CMV HaloTag®-LgBiT or CMV HaloTag®-HiBiT, using Lipofectamine 3000® according to the manufacturer's protocol for a 6-well transfection. For each transfection, use 2.5 µg (in total) of the linearized plasmid DNA.
5. 48 h after transfection, add Hygromycin B to the culture medium (200 μg/mL, Roche). Repeat the addition of the same amount of fresh Hygromycin B in the culture medium every 48 h until no living cells remain in the non-transfected control.
6. After completing the selection, expand the cells by further cultivation.

B. Preparation of pseudotyped alpharetroviral vectors for DF1 cells transduction with FuTraP genome

1. After the DF1 cells reach confluency between 80%–90%, discard the culture medium, wash cells with PBS and add prewarmed (37°C) trypsin-EDTA solution. Incubate the cells at 37°C for 5–8 min.
2. Resuspend the cells in the culture medium and determine the cell concentration.
3. For each transfection seed 4.5 × 10⁵ of DF1 cells on a 6-well plate. Cultivate the cells for 18–24 h.
4. Cotransfect the DF1 cells with 0.5 µg of pVSV-G (Clontech), 0.75 μg of pcGag-Pol (Plachý et al. 2010) and 1.25 μg of pFuTraP genome (Štafl et al. 2021), using Lipofectamine 3000® according to the manufacturer's protocol for a 6-well transfection. 
5. 24 h after transfection, discard the culture medium, 3× wash cells with addition of 2 mL of PBS and then add 2.5 mL of the fresh culture medium.
6. 72 h after transfection, collect the supernatant with a sterile needle and filter it through a 0.45 µm filter (Corning, NY).
7. Store the collected supernatants in -80°C.

C: Transduction of DF1/LgBiT cells with pseudotyped alpharetroviral vectors for ectopic human ASCT2 expression

1. After the DF1/LgBiT cells reach confluency between 80%–90%, discard the culture medium, wash the cells with PBS and add prewarmed (37°C) trypsin-EDTA solution. Incubate the cells at 37°C for 5–8 min.
2. Resuspend the cells in the culture medium and determine the cell concentration.
3. For each transduction, seed 1.5 × 10⁵ of DF1/LgBiT cells on a 6-well plate. Cultivate the cells for 18–24 h.
4. Wash the cells with PBS. For transduction, add 2 mL of the filtered supernatant containing pseudotyped viral particles with pFuTraP genome.
5. Let the transduced cells expand by further cultivation for about one week. Exchange the culture medium for a fresh one if needed. 
6. Proceed to FACS sorting for the expression of the iRFP713 reporter. It is recommended to repeat this step at least once due to increased confidence in the purity of the sorted cell population.
7. Expand the sorted DF1/LgBiT-FuTraP cells, now stably expressing LgBiT and the human ASCT2.

D: Cell-cell fusion assay

NOTE: It is possible to directly mix and co-cultivate DF1/HiBiT-pMCAS(Sync1-MSC16)dsRed cells with DF1/LgBiT-FuTraP cells on a white 96-well plate. However, this format allows co-cultivation of only 30,000 DF1 cells, and the luminescent signal originating from cell-cell fusion is therefore low.

1. After the DF1/HiBiT cells reach confluency between 80%–90%, discard the culture medium, wash cells with PBS and add prewarmed (37°C) trypsin-EDTA solution. Incubate the cells at 37°C for 5–8 min.
2. Resuspend the cells in the culture medium and determine the cell concentration.
3. For each transfection, seed 4.5 × 10⁵ of DF1/HiBiT cells on a 6-well plate. Cultivate the cells for 18–24 h.
4. Transfect the DF1/HiBiT cells with pMCAS(Sync1-MSC16)dsRed plasmid, using Lipofectamine 3000® according to the manufacturer's protocol for 6-well transfection. For each transfection, use 2.5 µg (in total) of plasmid DNA.
5. 48 h after transfection, detouch non-enzymaticaly the DF1/LgBiT-FuTraP cells and the transfected DF1/HiBiT-pMCAS(Sync1-MSC16)dsRed cells from the plastic by addition of 1× Cell Dissociation Solution (non-enzymatic; Sigma). Incubate the cells at 37°C for 5–10 min.
6. Resuspend the cells in the culture medium and determine the cell concentration.
7. Use part of the DF1/HiBiT-pMCAS(Sync1-MSC16)dsRed cells for flow cytometry analysis of dsRed expression.
8. Mix DF1/LgBiT-FuTraP cells and DF1/HiBiT-pMCAS(Sync1-MSC16)dsRed cells in ratio of 2 × 10⁵ : 4 × 10⁵ cells/well, respectively, on a 6-well plate. Add the culture medium to a total volume of 2 mL per well.
9. After 24 h of cultivation, wash cells with PBS and add prewarmed (37°C) trypsin-EDTA solution to detouch the cells from the plastic.
10. Stop the trypsin activity by addition of 1 mL culture medium. Transfer all the cells into 2mL microtube and centrifuge at 300 × g in 4 °C for 5 min.
11. Gently discard the supernatant and resuspend the cell pellet in 350 µl of OptiMEM.
12. Prepare the Nano-Glo® Live Cell Reagent (Promega) according to the manufacturer's protocol.
13. Transfer the resuspended cells per 100 µl (in technical triplicates) to a white 96-well plate (Costar) containing 20–25 µl of freshly prepared Nano-Glo® Live Cell Reagent (Promega). The background signal can be further reduced by adding the membrane-impermeable peptide DrkBiT peptide (Promega), which sequesters free LgBiT in the supernatant.
14. Incubate for 10 min at the room temperature. Shake the plate at 300 × rpm and measure the luminescence in a luminometer.