Table 1.

Dengue Virus Infection Assay Workflow

Step	Parameter	Value	Description
1	Cell plating	35 μL	4,000 HEK293 cells in inoculation MEM media
2	Incubation time	12 h	37°C, 5% CO2
3	Library compounds	5 μL	100 μM in 10% DMSO
4	Low control	5 μL	40 μg/mL carrageenan in water
5	High control	5 μL	10% DMSO
6	Virus Infection	10 μL	Addition of DENV-2 at an MOI of 0.5
7	Incubation time	48 h	37°C, 5% CO2
8	Fix	50 μL	4% PFA for 20 min
9	Wash	50 μL	Two times with 1 × PBS
10	Blocking buffer	50 μL	2% NGS, 0.4% TX-100 in 1 × PBS for 1 h
11	α-E primary staining	50 μL	1:1,000 dilution in 1 × PBS for 1 h
12	Wash buffer	50 μL	Three times with 0.2% BSA, 0.2% TX-100 in 1 × PBS
13	Detection secondary staining	50 μL	1:1,000 dilution of Alexa-Fluor 488 in 1 × PBS for 1 h
14	Wash buffer	50 μL	Three times with 0.2% BSA, 0.2% TX-100 in 1 × PBS
15	Nuclear staining	50 μL	10 μM Hoechst, 0.05% TX-100 in 1 × PBS for 10 min
16	Assay readout	364 nm/450 nm and 488 nm/535 nm (ex/em)	INCA3000 microscope system
17	Image analysis	—	Multiparametric analysis using Raven 1.0 software

Step Notes

1. Cells are prepared in media and dispensed into a 384-well assay plate with Multidrop 384

2. The 384-well assay plates are stored in the Steri-Cult, an automated, temperature-controlled incubator

3. to 5. Dispensing using a custom-designed 384-head on a PP-384-M Personal Pipettor; 30 s per 384-well microplate

6. Virus prepared in media and dispensed into 384-well assay plate with Multidrop 384

7. The 384-well assay plates are stored in the Steri-Cult

8. to 15. Dispensing and aspirating on the ELx405 washer; 1 min per 384-well microtiter plate

16. Readout performed on an automated platform; 14 s per well, with a total imaging time of 90 min per 384-well microtiter plate

17. Analysis of DENV E and Hoechst-stained nuclei; 20 min per 384-well microtiter plate

 BSA, bovine serum albumin; DENV, Dengue virus; DMSO, dimethyl sulfoxide; INCA3000, IN CELL Analyzer 3000; MOI, multiplicity of infection; PBS, phosphate-buffered saline.