High-Throughput Bioscience Center (HTBC) • Access to rapidly test thousands of molecules in various bioassays. – – Drug Repurposing Libraries— 8, 770 compounds of known modulators/FDA approved Drugs (LOPAC, NIHCC, NCI DTP, Micorsource, Biomol ICCB and FDA) – Small interfering RNAs (si. RNAs)—Whole genomes targeting 21, 000 genes, pools of 4 si. RNAs/gene, Human library from Dharmacon, and Mouse library from Qiagen – • Small Molecule Compound library— 114, 000 diverse compounds (Chem. Div, Chembridge, Specs, Chem. Div Kinase) c. DNAs— 15, 000 genes, E. coli stocks Human ORFeome in Gateway vectors from Open Biosystems High-Throughput Assays (384 -well plates) • Fully automated integrated system: In vitro targets--Enzymes, Protein-protein interactions, Ligand-receptor binding, Protein. DNA binding, etc. (10 -15, 000 compounds/day) – – In vivo cell based-spectrophotometric-Reporter gene-luciferase, proliferation, synthetic lethality, etc. (15 -30, 000 compounds per week) – – – High-Content Imaging--Mitosis, protein translocation/expression, cellular morphology, apoptosis, (5 -10, 000 compounds per week) – – 8, 96 or 384 tip pipettors/pin tools (Caliper. LS Sci. Clone ALH 3000) Robotic arms/Stackers for moving microplates (Zymark Twister II) Robotic 198 plate CO 2 Incubator Integrated plate washer/reagent dispenser (Bio-tek EL 406) and reagent dispenser (Multidrop) Bar Code reader Microplate reader (Molecular Devices Analyst. GT, Tecan Infinite M 1000)
si. RNA Screening at the HTBC • si. ARRAY whole Human Genome si. RNA library from Thermo. Fisher (formerly Dharmacon) – 80 gene targets per plate • • – 4 si. RNA oligos per gene--pooled in each well 267 X 96 well plates targeting 21, 122 genes Library subdivided in: • • 7 GPCR (517 genes) • 76 Druggable genome (6022 genes) • – 10 Kinase (779 genes, can be tested separately) 174 Rest of genome including XMs (13, 804 genes) All individual si. RNA oligos available for retests of hits • 4 individual oligos per gene • High-Throughput si. RNA assays performed in 96 - or 384 -well plates (384 -well preferred) • Mouse si. RNA library from Qiagen formatted in 384 -well plates. • Agilent system (96/384 well) – – – 6 stackers Bravo 96 or 384 tip pipetting head- 9 plate positions Thermal Plate Sealer Wellmate Reagent Dispenser (96/384 well) ELx 405 Plate Washer (96/384 well)
High-Content Cellular Imaging • Image. Xpress Micro (Molecular Devices) – Fully automated inverted epifluorescence microscope scans 6 - to 384 -well plates and records multiple fluorescent images – Live cell option-Temp, CO 2, humidity – Xenon Lamp/filter based – Complete spectral range with 15 filter set combinations – Up to four color imaging/any fluorophore – Phase Contrast and Brightfield • Robotic Arm Integration – – • • More Info: http: //htbc. stanford. edu David E. Solow-Cordero, Ph. D. Director, HTBC, CCSR-0133 desolow@stanford. edu 3 stackers for fixed plates Automated incubator for imaging up to 42 livecell plates Image Analysis Software – Meta. Xpress 5 with 10 Analysis modules – 2 analysis computers – 3 rd party/custom/ Matlab, Cell Profiler • Completed fully automated whole genome si. RNA High Content Screen – 200 X 384 -well plates imaged, 1 site, 4 colors in 10 weeks; 307, 000 images!
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