Table 1.

Protocol Outline for Plate Preparation with Automation

Sequence	Parameter	Value	Description
1	Reagent	5 µL	1000 cells/well in 1536    TC-treated plate
2	Time	18–20 h	37 °C 5% CO2
3	Reagent	23 nL	Compounds
4	Time	18 h	37 °C 5% CO2
5	Reagent	4 µL	6% PFA with 0.1% Triton X-100
6	Time	20 min	Room temperature—fume hood
7	Decant	8 µL	Kalypsys pipettor removes    media + PFA
8	Reagent	5 µL	HBSS buffer
9	Decant	4 µL	Kalypsys pipettor with HBSS
10	Reagent	5 µL	HBSS buffer
11	Decant	4 µL	Kalypsys pipettor removes HBSS
12	Reagent	5 µL	HBSS buffer with 10 µg/mL    Hoechst 33342
13	Detector	GFP filter set	IN Cell Analyzer 1000    measurement

GFP, green fluorescent protein; HBSS, Hank’s buffered salt solution; PFA, paraformaldehyde; TC, tissue culture.