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- incubationa/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-NMQa 1 | 250 mM sucrose, 10 mM TrisHCl, 10 mM MgCl2 | 5/2 | Yes | N/A |
pH 7.0, +/− 4 mM ATP, 1% DMSO | |||||||||||
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) |
40 Solvo | pH 7.4 with 4 mM ATP, ATP with 5 µM LSN335984, or 4 mM AMP-PNP, 0.5% DMSO | ||||||||||
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 |
pH 7.0, +/− 4 mM ATP, 1% DMSO | |||||||||||
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 |
+/− 4 mM ATP, 1% DMSO | |||||||||||
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.