High-Throughput Multiplexed Quantitation of Protein Aggregation and Cytotoxicity in a Huntington’s Disease Model

Table 1: Protocol for Multiplexed Protein Aggregation and Cytotoxicity Assay in 1536-Well Plate Format

Step	Parameter	Value	Description
1	Cell plating	5 µl/well	1,000cells/well
2	Incubation	24 hr	at 37 °C with 5% CO₂
3	Compound addition	0.023 µl/well	Compounds in DMSO solution and 200 nM Tebufenozide (inducer)
4	Incubation	48 hr	at 37 °C and 5% CO₂
5	Reagent-1	2 µl/well	reagent mixture from the protease assay kit
6	Incubation	10 min	at 37 °C
7	Plate reading-1	luminescence	Viewlux plate reader
8	Reagent-2 (cell lysis)	2 µl/well	0.25% Triton X-100 (final)
9	Incubation	30 min	room temperature
10	Plate reading-2	Ex=488 nm, Em= 500-530nm	Acumen Explore reader to count # of aggregates