By Hao Zhu, Menghang Xia
This booklet makes a speciality of lately built excessive Throughput Screening (HTS) assay protocols, many focused on the ToxCast and/or Tox21 projects, and the correct HTS facts research suggestions. Divided into 3 sections, in vitro assays, in vivo assays, and computational recommendations to research HTS information are all tested. Written for the hugely profitable Methods in Molecular Biology sequence, so much chapters contain introductions to their respective themes, lists of the required fabrics and reagents, step by step, effectively reproducible laboratory protocols, and tips about troubleshooting and warding off identified pitfalls.
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Extra resources for High-Throughput Screening Assays in Toxicology
6. 75 % bovine serum albumin (BSA) in Phosphate Buffered Saline (PBS) buffer. 7. Mantis single-channel low (Formulatrix, Bedford, MA). volume liquid dispenser 8. Pintool workstation (Wako Automation, San Diego, CA). 9. Plate centrifuge and microcentrifuge. 10. ThermoMixer C (Eppendorf North America, Hauppauge, NY). 11. Envision plate reader with a LANCE/DEFIA D400/630 dual mirror, an excitation filter of 340/60 nm, and emission filters of 495/10 nm and 520/25 nm (PerkinElmer, Waltham, MA). 1 Methods Cell Thawing 1.
2. The cells were plated at 2000 cells/well in 5 μL of the culture medium into a 1536-well black wall/clear bottom plate using Multidrop Combi (see Note 1). 3. The assay plates were incubated overnight at 37 °C for cell adhesion. 4. The adhered cells were treated with 23 nL of test compounds and positive control using a Pintool. 5 μM FCCP, respectively), and DMSO only was transferred to column 4. 5. The assay plates were incubated at 37 °C for 1 h or 5 h. 6. After the respective incubation times, 5 μL of 2x m-MPI dyeloading solution (10 μL of m-MPI stock solution added to 5 mL of MMP assay buffer, mixed well by vortexing) was added to each well using FRD (see Note 2).
10. Envision bottom mirror: Beta-Lactamase Dual Enh. D425 nm/D490 nm #661, excitation filter: Photometric 405 nm, emission filter: FITC 535 nm and second emission filter: Umbelliferone 460 nm (PerkinElmer, Waltham, MA). 1 Cell Line and Culture Condition 1. X-Man® HIF1A NanoLuc HCT116 protein reporter cell line (Horizon Discovery, Cambridge, UK). 2. Thaw, Cell Culture, and Assay Medium: RPMI 1640 medium supplement with 10 % Hyclone defined fetal bovine serum, 100U/mL penicillin and 100 μg/mL streptomycin, and 300 μg/mL G418.