TABLE 4

Experimental details by company (Vesicles)

Lab ID	Vesicle Source	Filtration Method (Vac. Manifold, Cell Harvester, etc.)	Protein/Well	Assay Temperature	Filter Plate Vendor Plus Cat. No.	Signal-to-Noise Acceptance Criteria	Substrate and Conc.	Assay Buffer and Solvent Content	Pre- incubation^{^a}/Assay Time	Plate Shaking	Positive Control Inhibitor
			mg				μM		min	rpm	conc.
23	In-house	Vacuum manifold (Millipore)	50	37°C	Millipore, MSFBN6B50, MultiScreen HTS96-well, GF/B, 1.0 μm	>6-Fold difference between ATP+ and ATP–	³H-NMQ^{^a} 1	250 mM sucrose, 10 mM TrisHCl, 10 mM MgCl₂	5/2	Yes	N/A
23	In-house	Vacuum manifold (Millipore)	50	37°C	Millipore, MSFBN6B50, MultiScreen HTS96-well, GF/B, 1.0 μm	>6-Fold difference between ATP+ and ATP–	³H-NMQ^{^a} 1	pH 7.0, +/− 4 mM ATP, 1% DMSO	5/2	Yes	N/A
26	In-house and Solvo	96-Channel cell harvester (PerkinElmer)	20 Lilly	37°C	PerkinElmer; 600517796-well GF/B, 1.0 μm	AMP-PNP value = inhibitor control	³H Vinblastine 0.1	250 mM sucrose, 10 mM TrisHCl, 10 mM MgCl₂	10/2.5	Yes	LSN335984, (5µM)
26	In-house and Solvo	96-Channel cell harvester (PerkinElmer)	40 Solvo	37°C	PerkinElmer; 600517796-well GF/B, 1.0 μm	AMP-PNP value = inhibitor control	³H Vinblastine 0.1	pH 7.4 with 4 mM ATP, ATP with 5 µM LSN335984, or 4 mM AMP-PNP, 0.5% DMSO	10/2.5	Yes	LSN335984, (5µM)
22	Solvo	Vacuum manifold (Millipore)	50	37°C	Millipore, MSFBN6B10, MultiScreen 96-well, GF/B, 1.0 μm	>3-Fold difference between ATP+ and ATP-	NMQ 2	250 mM sucrose, 10 mM TrisHCl, 10 mM MgCl₂	15/3	No	Ketoconazole 8 pt IC₅₀
22	Solvo	Vacuum manifold (Millipore)	50	37°C	Millipore, MSFBN6B10, MultiScreen 96-well, GF/B, 1.0 μm	>3-Fold difference between ATP+ and ATP-	NMQ 2	pH 7.0, +/− 4 mM ATP, 1% DMSO	15/3	No	Ketoconazole 8 pt IC₅₀
21	Solvo	Vacuum manifold (Millipore)	50	37°C	Millipore, MSFBN6B50, MultiScreen HTS 96-well, GF/B, 1.0 μm	>3-Fold difference between ATP+ and ATP-	³H-NMQ 1	Assay buffer (and all other buffers) from PREDIVEZ Vesicular Transport Kit (SOLVO)	15/2	Yes	Vinblastine or erythromycin 4 pt IC₅₀
21	Solvo	Vacuum manifold (Millipore)	50	37°C	Millipore, MSFBN6B50, MultiScreen HTS 96-well, GF/B, 1.0 μm	>3-Fold difference between ATP+ and ATP-	³H-NMQ 1	+/− 4 mM ATP, 1% DMSO	15/2	Yes	Vinblastine or erythromycin 4 pt IC₅₀
20	Solvo	Vacuum manifold (Millipore)	50	37°C	Millipore, MSFBN6B50, MultiScreen HTS 96-well, GF/B, 1.0 μm	>6-Fold difference between ATP+ and ATP–	³H-NMQ 2	250 mM sucrose, 10 mM TrisHCl, 10 mM MgCl₂pH 7.0, +/− 4 mM ATP, 1% DMSO	15/3	No	N/A

For abbreviations, see Table 1.
↵a NMQ, N-methylquinidine.