Table 1.

Protocol for Virus–Cell Fusion High-Throughput Screening Assay in Regular 384-Well Cell Culture Plate

Step	Parameter	Value	Description
1	Plate cells	25 μL	20,000 TZM-bl cells
2	Incubation time	24 h	37°C, 5% CO2
3	Add virus	5 μL	HIV-1 pseudoviruses at MOI 1.0
4	Centrifuge	30 min	2,095 g at 4°C
5	Library compound	0.1 μL	The final compound concentration is 10 μM
6	Add positive control	0.5 μL	Positive control C52L peptide diluted in medium added at final concentration of 1 μM
7	Incubation time	90 min	37°C, 5% CO2
8	Add CCF4-AM substrate	25 μL	Remove the medium before adding substrate
9	Incubation time	16 h	Overnight incubation at 12°C
10	Assay readout	Excitation at 400 nm, emissions at 460 nm and 520 nm	EnVision Multilabel plate reader

Step Notes

1. Black wall clear-bottom cell culture plate was used. Cells were dispensed to all wells.

3. Virus was added from column 2 to 24. Equal volume of medium was added to column 1 as no virus control. MOI is multiplicity of infection.

5. Pintool was used for compound transfer.

6. Positive control C52L was added to column 24.

8. Follow manufacturer's instruction for preparing substrate. Substrate was added from column 3 to column 24.

10. Bottom read module was used for reading plate.