Table 3.

High-Content Imaging Analysis of Cytotoxicity in Induced Pluripotent Stem Cell–Endothelial Cells and Human Umbilical Vein Endothelial Cells

Step	Parameter	Value	Description
1	Dispense fibronectin solution	10 μL	30 μg/mL fibronectin in water
2	Incubation time	2 h	Room temperature
3	Plate cells	25 μL	7.5 × 102
4	Incubation time	5–6 days	37°C, 5% CO2
5	Change media	25 μL	Every other day
6	Library compounds	12.5 μL	4 × concentration stock
7	Incubation time	1 h	37°C, 5% CO2
8	Cytokine stimulation (optional)	12.5 μL	A cocktail of cytokines (IL-1β, 1 ng/mL; TNF-α, 5 ng/mL; and IFN-γ, 100 ng/mL) in maintenance medium.
9	Incubation time	24 h	37°C, 5% CO2
10	Dispense 4 × staining solution	16.7 μL	With HBSS
11	Incubation time	20 min	Room temperature
12	Wash	2 Times	With HBSS
13	Dispense HBSS	25 μL
14	Acquire images	10 × Objective	With DAPI, FITC filter
15	Dispense CellTiter-Glo® solution	25 μL	CellTiter-Glo Luminescent Cell Viability Assay
16	Incubation time	10 min	Room temperature
17	Reading	Luminescence	FLIPR Tetra®

Step Notes

1, 2. These steps are for iCell endothelial cells.

3. Plate for iCell endothelial cells and HUVECs: black clear-bottom 384-well plate. iCell endothelial cells: remove fibronectin solution before plating cells.

5. For cell maintenance, 25 μL of medium were exchanged every other day. Cell maintenance continued until the monolayer was formed. On the evening before the experiment, old medium was replaced with 25 μL fresh medium.

8. For nonstimulated cells, 12.5 μL/well normal maintenance medium was dispended.

14. ImageXpress was used for image acquisition.

 HBSS, Hank's balanced buffer solution.